Method of Determining the Nucleotide Sequence of Oligonucleotides and DNA Molecules

ABSTRACT

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of Ser. No. 11/929,141 filedOct. 30, 2007, which is a continuation of Ser. No. 10/709,436 filed May5, 2004, now U.S. Pat. No. 7,645,596, which is a continuation of Ser.No. 09/941,882 filed Aug. 28, 2001, now U.S. Pat. No. 6,780,591, whichis a continuation-in-part of Ser. No. 09/673,544 filed Feb. 26, 2001 andnow abandoned, which is a continuation-in-part of PCT/US99/09616 filedApr. 30, 1999 and claims the benefit of provisional application Ser. No.60/083,840 filed May 1, 1998.

INTRODUCTION

The present invention relates to a novel method for analyzing nucleicacid sequences based on real-time detection of DNA polymerase-catalyzedincorporation of each of the four deoxynucleoside monophosphates,supplied individually and serially as deoxynucleoside triphosphates in amicro fluidic system, to a template system comprising a DNA fragment ofunknown sequence and an oligonucleotide primer. Incorporation of adeoxynucleoside monophosphate (dNMP) into the primer can be detected byany of a variety of methods including but not limited to fluorescenceand chemiluminescence detection. Alternatively, microcalorimeticdetection of the heat generated by the incorporation of a dNMP into theextending primer using thermopile, thermistor and refractive indexmeasurements can be used to detect extension reactions. The presentinvention further provides a method for monitoring and correction ofsequencing errors due to misincorporation or extension failure.

The present invention provides a method for sequencing DNA that avoidselectrophoretic separation of DNA fragments thus eliminating theproblems associated with anomalous migration of DNA due to repeated basesequences or other self-complementary sequences which can causesingle-stranded DNA to self-hybridize into hairpin loops, and alsoavoids current limitations on the size of fragments that can be read.The method of the invention can be utilized to determine the nucleotidesequence of genomic or cDNA fragments, or alternatively, as a diagnostictool for sequencing patient derived DNA samples.

BACKGROUND OF INVENTION

Currently, two approaches are utilized for DNA sequence determination:the dideoxy chain termination method of Sanger (1977, Proc. Natl. Acad.Sci. 74:5463-5674) and the chemical degradation method of Maxam (1977,Proc. Natl. Acad. Sci. 74:560-564). The Sanger dideoxy chain terminationmethod is the most widely used method and is the method upon whichautomated DNA sequencing machines rely. In the chain termination method,DNA polymerase enzyme is added to four separate reaction systems to makemultiple copies of a template DNA strand in which the growth process hasbeen arrested at each occurrence of an A, in one set of reactions, and aG, C, or T, respectively, in the other sets of reactions, byincorporating in each reaction system one nucleotide type lacking the3′-OH on the deoxyribose at which chain extension occurs. This procedureproduces a series of DNA fragments of different lengths, and it is thelength of the extended DNA fragment that signals the position along thetemplate strand at which each of four bases occur. To determine thenucleotide sequence, the DNA fragments are separated by high resolutiongel electrophoresis and the order of the four bases is read from thegel.

A major research goal is to derive the DNA sequence of the entire humangenome. To meet this goal the need has developed for new genomicsequencing technology that can dispense with the difficulties of gelelectrophoresis, lower the costs of performing sequencing reactions,including reagent costs, increase the speed and accuracy of sequencing,and increase the length of sequence that can be read in a single step.Potential improvements in sequencing speed may be provided by acommercialized capillary gel electrophoresis technique such as thatdescribed in Marshall and Pennisis (1998, Science 280:994-995). However,a major problem common to all gel electrophoresis approaches is theoccurrence of DNA sequence compressions, usually arising from secondarystructures in the DNA fragment, which result in anomalous migration ofcertain DNA fragments through the gel.

As genomic information accumulates and the relationships between genemutations and specific diseases are identified, there will be a growingneed for diagnostic methods for identification of mutations. In contrastto the large scale methods needed for sequencing large segments of thehuman genome, what is needed for diagnostic methods are repetitive,low-cost, highly accurate techniques for resequencing of certain smallisolated regions of the genome. In such instances, methods of sequencingbased on gel electrophoresis readout become far too slow and expensive.

When considering novel DNA sequencing techniques, the possibility ofreading the sequence directly, much as the cell does, rather thanindirectly as in the Sanger dideoxynucleotide approach, is a preferredgoal. This was the goal of early unsuccessful attempts to determine theshapes of the individual nucleotide bases with scanning probemicroscopes.

Additionally, another approach for reading a nucleotide sequencedirectly is to treat the DNA with an exonuclease coupled with adetection scheme for identifying each nucleotide sequentially releasedas described in Goodwin, et al., (1995, Experimental Techniques ofPhysics 41:279-294). However, researchers using this technology areconfronted with the enormous problem of detecting and identifying singlenucleotide molecules as they are digested from a single DNA strand.Simultaneous exonuclease digestion of multiple DNA strands to yieldlarger signals is not feasible because the enzymes rapidly get out ofphase, so that nucleotides from different positions on the differentstrands are released together, and the sequences become unreadable. Itwould be highly beneficial if some means of external regulation of theexonuclease could be found so that multiple enzyme molecules could becompelled to operate in phase. However, external regulation of an enzymethat remains docked to its polymeric substrate is exceptionallydifficult, if not impossible, because after each digestion the nextsubstrate segment is immediately present at the active site. Thus, anycontrolling signal must be present at the active site at the start ofeach reaction.

A variety of methods may be used to detect the polymerase-catalyzedincorporation of deoxynucleoside monophosphates (dNMPs) into a primer ateach template site. For example, the pyrophosphate released whenever DNApolymerase adds one of the four dNTPs onto a primer 3′ end may bedetected using a chemiluminescent based detection of the pyrophosphateas described in Hyman E. D. (1988, Analytical Biochemistry 174:423-436)and U.S. Pat. No. 4,971,903. This approach has been utilized mostrecently in a sequencing approach referred to as “sequencing byincorporation” as described in Ronaghi (1996, Analytical Biochem.242:84) and Ronaghi (1998, Science 281:363-365). However, there existtwo key problems associated with this approach, destruction ofunincorporated nucleotides and detection of pyrophosphate. The solutionto the first problem is to destroy the added, unincorporated nucleotidesusing a dNTP-digesting enzyme such as apyrase. The solution to thesecond is the detection of the pyrophosphate using ATP sulfurylase toreconvert the pyrophosphate to ATP which can be detected by a luciferasechemiluminescent reaction as described in U.S. Pat. No. 4,971,903 andRonaghi (1998, Science 281:363-365). Deoxyadenosine a-thiotriphosphateis used instead of dATP to minimize direct interaction of injected dATPwith the luciferase.

Unfortunately, the requirement for multiple enzyme reactions to becompleted in each cycle imposes restrictions on the speed of thisapproach while the read length is limited by the impossibility ofcompletely destroying unincorporated, non-complementary, nucleotides. Ifsome residual amount of one nucleotide remains in the reaction system atthe time when a fresh aliquot of a different nucleotide is added for thenext extension reaction, there exists a possibility that some fractionof the primer strands will be extended by two or more nucleotides, theadded nucleotide type and the residual impurity type, if these match thetemplate sequence, and so this fraction of the primer strands will thenbe out of phase with the remainder. This out of phase component producesan erroneous incorporation signal which grows larger with each cycle andultimately makes the sequence unreadable.

A different direct sequencing approach uses dNTPs tagged at the 3′ OHposition with four different colored fluorescent tags, one for each ofthe four nucleotides is described in Metzger, M. L., et al. (1994,Nucleic Acids Research 22:4259-4267). In this approach, theprimer/template duplex is contacted with all four dNTPs simultaneously.Incorporation of a 3′ tagged NMP blocks further chain extension. Theexcess and unreacted dNTPs are flushed away and the incorporatednucleotide is identified by the color of the incorporated fluorescenttag. The fluorescent tag must then be removed in order for a subsequentincorporation reaction to occur. Similar to the pyrophosphate detectionmethod, incomplete removal of a blocking fluorescent tag leaves someprimer strands unextended on the next reaction cycle, and if these aresubsequently unblocked in a later cycle, once again an out-of-phasesignal is produced which grows larger with each cycle and ultimatelylimits the read length. To date, this method has so far beendemonstrated to work for only a single base extension. Thus, this methodis slow and is likely to be restricted to very short read lengths due tothe fact that 99% efficiency in removal of the tag is required to readbeyond 50 base pairs. Incomplete removal of the label results in out ofphase extended DNA strands.

SUMMARY OF INVENTION

Accordingly, it is an object of the present invention to provide a novelmethod for determining the nucleotide sequence of a DNA fragment whicheliminates the need for electrophoretic separation of DNA fragments. Theinventive method, referred to herein as “reactive sequencing”, is basedon detection of DNA polymerase catalyzed incorporation of each of thefour nucleotide types, when deoxynucleoside triphosphates (dNTP's) aresupplied individually and serially to a DNA primer/template system. TheDNA primer/template system comprises a single stranded DNA fragment ofunknown sequence, an oligonucleotide primer that forms a matched duplexwith a short region of the single stranded DNA, and a DNA polymeraseenzyme. The enzyme may either be already present in the template system,or may be supplied together with the dNTP solution.

Typically a single deoxynucleoside triphosphate (dNTP) is added to theDNA primer template system and allowed to react. As used hereindeoxyribonucleotide means and includes, in addition to dGTP, dCTP, dATP,dTTP, chemically modified versions of these deoxyribonucleotides oranalogs thereof. Such chemically modified deoxyribonucleotides includebut are not limited to those deoxyribonucleotides tagged with afluorescent or chemiluminescent moiety. Analogs of deoxyribonucleotidesthat may be used include but are not limited to 7-deazapurine. Thepresent invention additionally provides a method for improving thepurity of deoxynucleotides used in the polymerase reaction.

An extension reaction will occur only when the incoming dNTP base iscomplementary to the next unpaired base of the DNA template beyond the3′ end of the primer. While the reaction is occurring, or after a delayof sufficient duration to allow a reaction to occur, the system istested to determine whether an additional nucleotide derived from theadded dNTP has been incorporated into the DNA primer/template system. Acorrelation between the dNTP added to the reaction cell and detection ofan incorporation signal identifies the nucleotide incorporated into theprimer/template. The amplitude of the incorporation signal identifiesthe number of nucleotides incorporated, and thereby quantifies singlebase repeat lengths where these occur. By repeating this process witheach of the four nucleotides individually, the sequence of the templatecan be directly read in the 5′ to 3′ direction one nucleotide at a time.

Detection of the polymerase mediated extension reaction andquantification of the extent of reaction can occur by a variety ofdifferent techniques, including but not limited to, microcalorimeticdetection of the heat generated by the incorporation of a nucleotideinto the extending duplex. Optical detection of an extension reaction byfluorescence or chemiluminescence may also be used to detectincorporation of nucleotides tagged with fluorescent or chemiluminescententities into the extending duplex. Where the incorporated nucleotide istagged with a fluorophore, excess unincorporated nucleotide is removed,and the template system is illuminated to stimulate fluorescence fromthe incorporated nucleotide. The fluorescent tag may then be cleaved andremoved from the DNA template system before a subsequent incorporationcycle begins. A similar process is followed for chemiluminescent tags,with the chemiluminescent reaction being stimulated by introducing anappropriate reagent into the system, again after excess unreacted taggeddNTP has been removed; however, chemiluminescent tags are typicallydestroyed in the process of readout and so a separate cleavage andremoval step following detection may not be required. For either type oftag, fluorescent or chemiluminescent, the tag may also be cleaved afterincorporation and transported to a separate detection chamber forfluorescent or chemiluminescent detection. In this way, fluorescentquenching by adjacent fluorophore tags incorporated in a single baserepeat sequence may be avoided. In addition, this may protect the DNAtemplate system from possible radiation damage in the case offluorescent detection or from possible chemical damage in the case ofchemiluminescent detection. Alternatively the fluorescent tag may beselectively destroyed by a chemical or photochemical reaction. Thisprocess eliminates the need to cleave the tag after each readout, or todetach and transport the tag from the reaction chamber to a separatedetection chamber for fluorescent detection. The present inventionprovides a method for selective destruction of a fluorescent tag by aphotochemical reaction with diphenyliodonium ions or related species.

The present invention further provides a reactive sequencing method thatutilizes a two cycle system. An exonuclease-deficient polymerase is usedin the first cycle and a mixture of exonuclease-deficient andexonuclease-proficient enzymes are used in the second cycle. In thefirst cycle, the template-primer system together with an exonucleasedeficient polymerase will be presented sequentially with each of thefour possible nucleotides. In the second cycle, after identification ofthe correct nucleotide, a mixture of exonuclease proficient anddeficient polymerases, or a polymerase containing both types of activitywill be added in a second cycle together with the correct dNTPidentified in the first cycle to complete and proofread the primerextension. In this way, an exonuclease-proficient polymerase is onlypresent in the reaction cell when the correct dNTP is present, so thatexonucleolytic degradation of correctly extended strands does not occur,while degradation and correct re-extension of previously incorrectlyextended strands does occur, thus achieving extremely accurate strandextension.

The present invention also provides a method for monitoring reactivesequencing reactions to detect and correct sequencing reaction errorsresulting from misincorporation, i.e., incorrectly incorporating anon-complementary base, and extension failure, i.e., failure to extend afraction of the DNA primer strands. The method is based on the abilityto (i) determine the size of the trailing strand population (trailingstrands are those primer strands which have undergone an extensionfailure at any extension prior to the current reaction step); (ii)determine the downstream sequence of the trailing strand populationbetween the 3′ terminus of the trailing strands and the 3′ terminus ofthe corresponding leading strands (“downstream” refers to the templatesequence beyond the current 3′ terminus of a primer strand;correspondingly, “upstream” refers to the known template andcomplementary primer sequence towards the 5′ end of the primer strand;“leading strands” are those primer strands which have not previouslyundergone extension failure); and (iii) predict at each extension stepthe signal to be expected from the extension of the trailing strandsthrough simulation of the occurrence of an extension failure at anypoint upstream from the 3′ terminus of the leading strand. Subtractionof the predicted signal from the measured signal yields a signal dueonly to valid extension of the leading strand population.

In a preferred embodiment of the invention, the monitoring for reactivesequencing reaction errors is computer-aided. The ability to monitorextension failures permits determination of the point to which thetrailing strands for a given template sequence have advanced and thesequence in the 1, 2 or 3 base gap between these strands and the leadingstrands. Knowing this information the dNTP probe cycle can be altered toselectively extend the trailing strands for a given template sequencewhile not extending the leading strands, thereby resynchronizing thepopulations.

The present invention further provides an apparatus for DNA sequencingcomprising: (a) at least one chamber including a DNA primer/templatesystem which produces a detectable signal when a DNA polymerase enzymeincorporates a deoxyribonucleotide monophosphate onto the 3′ end of theprimer strand; (b) means for introducing into, and evacuating from, thereaction chamber at least one selected from the group consisting ofbuffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides,and polymerase enzymes; (c) means for amplifying said signal; and (d)means for, converting said signal into an electrical signal.

BRIEF DESCRIPTION OF DRAWINGS

Further objects and advantages of the invention will be apparent from areading of the following description in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic diagram illustrating a reactive sequencing devicecontaining a thin film bismuth antimony thermopile in accordance withthe invention;

FIG. 2 is a schematic diagram of a reactive sequencing device containinga thermistor in accordance with the invention;

FIG. 3 is a schematic diagram illustrating a representative embodimentof micro calorimetry detection of a DNA polymerase reaction inaccordance with the invention;

FIG. 4 is an electrophoretic gel showing a time course for primerextension assays catalyzed by T4 DNA polymerase mutants;

FIG. 5 is a schematic diagram illustrating a nucleotide attached to afluorophore by a benzoin ester which is a photocleavable linker for usein the invention;

FIG. 6 is a schematic illustration of a nucleotide attached to achemiluminescent tag for use in the invention;

FIG. 7 is a schematic diagram of a nucleotide attached to achemiluminescent tag by a cleavable linkage;

FIGS. 8( a) and 8(b) are schematic diagrams of a mechanical fluorescentsequencing method in accordance with the invention in which a DNAtemplate and primer are absorbed on beads captured behind a porous frit;and

FIG. 9 is a schematic diagram of a sequencing method in accordance withthe invention utilizing a two cycle system.

FIG. 10 is a diagram of the mechanism of photochemical degradation offluorescein by diphenyliodonium ion (DPI).

FIG. 11 shows fluorescence spectra of equimolar concentrations offluorescein and tetramethylrhodamine dyes before and after addition of asolution of diphenyliodonium chloride.

FIG. 12 is the UV absorption spectra obtained from (1) fluorescein and(2) fluorescein+DPI after a single flash from a xenon camera strobe.

FIG. 13 displays the fluorescence spectra from single nucleotidepolymerase reactions with DPI photobleaching between incorporationreactions.

FIG. 14A-D. Simulation of Reactive Sequencing of [CTGA] GAA ACC AGA AAGTCC [T] (SEQ ID NO: 1), probed with a dNTP cycle. 14A (SEQ ID NO: 7).Sequence readout close to the primer where no extension failure hasoccurred. 14B (SEQ ID NO: 7). Sequence readout downstream of primerwhere 60% of the strands have undergone extension failure and areproducing out of phase signals and misincorporation has preventedextension on 75% of all strands. 14C (SEQ ID NO: 7). Downstream readoutwith error signals from trailing strands (dark shading) distinguishedfrom correct readout signals from leading strands (light shading) usingknowledge of the downstream sequence of the trailing strands. 14D (SEQID NO: 7). Corrected sequence readout following subtraction of errorsignals from trailing strands. Note the similarity to the data of FIG.1A.

FIG. 15 (SEQ ID NO: 8). Effect of a leading strand population onextension signals.

DETAILED DESCRIPTION

The present invention provides a method for determining the nucleic acidsequence of a DNA molecule based on detection of successive singlenucleotide DNA polymerase mediated extension reactions. As described indetail below, in one embodiment, a DNA primer/template system comprisinga polynucleotide primer complementary to and bound to a region of theDNA to be sequenced is constrained within a reaction cell into whichbuffer solutions containing various reagents necessary for a DNApolymerase reaction to occur are added. Into the reaction cell, a singletype of deoxynucleoside triphosphate (dNTP) is added. Depending on theidentity of the next complementary site in the DNA primer/templatesystem, an extension reaction will occur only when the appropriatenucleotide is present in the reaction cell. A correlation between thenucleotide present in the reaction cell and detection of anincorporation signal identifies the next nucleotide of the template.Following each extension reaction, the reaction cell is flushed withdNTP-free buffer, retaining the DNA primer/template system, and thecycle is repeated until the entire nucleotide sequence is identified.

The present invention is based on the existence of a control signalwithin the active site of DNA polymerases which distinguish, with highfidelity, complementary and non-complementary fits of incomingdeoxynucleotide triphosphates to the base on the template strand at theprimer extension site, i.e., to read the sequence, and to incorporate atthat site only the one type of deoxynucleotide that is complementary.That is, if the available nucleotide type is not complementary to thenext template site, the polymerase is inactive, thus, the templatesequence is the DNA polymerase control signal. Therefore, by contactinga DNA polymerase system with a single nucleotide type rather than allfour, the next base in the sequence can be identified by detectingwhether or not a reaction occurs. Further, single base repeat lengthscan be quantified by quantifying the extent of reaction.

As a first step in the practice of the inventive method, single-strandedtemplate DNA to be sequenced is prepared using any of a variety ofdifferent methods known in the art. Two types of DNA can be used astemplates in the sequencing reactions. Pure single-stranded DNA such asthat obtained from recombinant bacteriophage can be used. The use ofbacteriophage provides a method for producing large quantities of puresingle stranded template. Alternatively, single-stranded DNA may bederived from double-stranded DNA that has been denatured by heat oralkaline conditions, as described in Chen and Subrung, (1985, DNA4:165); Huttoi and Skaki (1986, Anal. Biochem. 152:232); and Mierendorfand Pfeffer, (1987, Methods Enzymol. 152:556), may be used. Such doublestranded DNA includes, for example, DNA samples derived from patients tobe used in diagnostic sequencing reactions.

The template DNA can be prepared by various techniques well known tothose of skill in the art. For example, template DNA can be prepared asvector inserts using any conventional cloning methods, including thoseused frequently for sequencing. Such methods can be found in Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition (Cold SpringHarbor Laboratories, New York, 1989). In a preferred embodiment of theinvention, polymerase chain reactions (PCR) may be used to amplifyfragments of DNA to be used as template DNA as described in Innis etal., ed. PCR Protocols (Academic Press, New York, 1990).

The amount of DNA template needed for accurate detection of thepolymerase reaction will depend on the detection technique used. Forexample, for optical detection, e.g., fluorescence or chemiluminescencedetection, relatively small quantities of DNA in the femtomole range areneeded. For thermal detection quantities approaching one picomole may berequired to detect the change in temperature resulting from a DNApolymerase mediated extension reaction.

In enzymatic sequencing reactions, the priming of DNA synthesis isachieved by the use of an oligonucleotide primer with a base sequencethat is complementary to, and therefore capable of binding to, aspecific region on the template DNA sequence. In instances where thetemplate DNA is obtained as single stranded DNA from bacteriophage, oras double stranded DNA derived from plasmids, “universal” primers thatare complementary to sequences in the vectors, i.e., the bacteriophage,cosmid and plasmid vectors, and that flank the template DNA, can beused.

Primer oligonucleotides are chosen to form highly stable duplexes thatbind to the template DNA sequences and remain intact during any washingsteps during the extension cycles. Preferably, the length of the primeroligonucleotide is from 18-30 nucleotides and contains a balanced basecomposition. The structure of the primer should also be analyzed toconfirm that it does not contain regions of dyad symmetry which can foldand self anneal to form secondary structures thereby rendering theprimers inefficient. Conditions for selecting appropriate hybridizationconditions for binding of the oligonucleotide primers in the templatesystems will depend on the primer sequence and are well known to thoseof skill in the art.

In utilizing the reactive sequencing method of the invention, a varietyof diffe to incorporate dNTPs onto the 3′ end of the primer which ishybridized to the polymerases include but are not limited to Taqpolymerase, T7 or T4 polyme preferred embodiment of the invention,described in detail below, DNA polyn proofreading activity are used inthe sequencing reactions. For the most rapi polymerase is sufficient toensure that each DNA molecule carries a non-co % molecule duringreaction. For a typical equilibrium constant of −50 nM for theequilibrium:

DNA-Pol DNA+Pol K

-   -   The desired condition is [Pol]≧50 nM+[DNA].

In addition, reverse transcriptase which catalyzes the synthesis ofsingle stranded DNA from an RNA template may be utilized in the reactivesequencing method of the invention to sequence messenger RNA (mRNA).Such a method comprises sequentially contacting an RNA template annealedto a primer (RNA primer/template) with dNTPs in the presence of reversetranscriptase enzyme to determine the sequence of the RNA. Because mRNAis produced by RNA polymerase-catalyzed synthesis from a DNA template,and thus contains the sequence information of the DNA template strand,sequencing the mRNA yields the sequence of the DNA gene from which itwas transcribed. Eukaryotic mRNAs have poly(A) tails and therefore theprimer for reverse transcription can be an oligo(dT). Typically, it willbe most convenient to synthesize the oligo(dT) primer with a terminalbiotin or amino group through which the primer can be captured on asubstrate and subsequently hybridize to and capture the template mRNAstrand.

The extension reactions are carried out in buffer solutions whichcontain the appropriate concentrations of salts, dNTPs and DNApolymerase required for the DNA polymerase mediated extension toproceed. For guidance regarding such conditions see, for example,Sambrook, et al., (1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.); and Ausubel, et al (1989, Current Protocolsin Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.).

Typically, buffer containing one of the four dNTPs is added into areaction cell. Depending on the identity of the nucleoside base at thenext unpaired template site in the primer/template system, a reactionwill occur when the reaction cell contains the appropriate dNTP. Whenthe reaction cell contains any one of the other three incorrect dNTPs,no reaction will take place.

The reaction cell is then flushed with dNTP free buffer and the cycle isrepeated until a complete DNA sequence is identified. Detection of a DNApolymerase mediated extension can be made using any of the detectionmethods described in detail below including optical and thermaldetection of an extension reaction.

In some instances, a nucleotide solution is found to be contaminatedwith any of the other three nucleotides. In such instances a smallfraction of strands may be extended by incorporation of an impurity dNTPwhen the dNTP type supplied is incorrect for extension, producing apopulation of strands which are subsequently extended ahead of the mainstrand population. Thus, in an embodiment of the invention, eachnucleotide solution can be treated to remove any contaminatednucleotides. Treatment of each nucleotide solution involves reaction ofthe solution prior to use with immobilized DNA complementary to each thepossibly contaminating nucleotides. For example, a dATP solution will beallowed to react with immobilized poly (dA), poly (dG) or poly (dC),with appropriate primers and polymerase, for a time sufficient toincorporate any contaminating dTTP, dCTP and dGTP nucleotides into DNA.

In a preferred embodiment of the invention, the primer/template systemcomprises the template DNA tethered to a solid phase support to permitthe sequential addition of sequencing reaction reagents withoutcomplicated and time consuming purification steps following eachextension reaction. Preferably, the template DNA is covalently attachedto a solid phase support, such as the surface of a reaction flow cell, apolymeric microsphere, filter material, or the like, which permits thesequential application of sequencing reaction reagents, i.e., buffers,dNTPs and DNA polymerase, without complicated and time consumingpurification steps following each extension reaction. Alternatively, forapplications that require sequencing of many samples containing the samevector template or same gene, for example, in diagnostic applications, auniversal primer may be tethered to a support, and the template DNAallowed to hybridize to the immobilized primer.

The DNA may be modified to facilitate covalent or non-covalent tetheringof the DNA to a solid phase support. For example, when PCR is used toamplify DNA fragments, the 5′ ends of one set of PCR primeroligonucleotides strands may be modified to carry a linker moiety fortethering one of the two complementary types of DNA strands produced toa solid phase support. Such linker moieties include, for example,biotin. When using biotin, the biotinylated DNA fragments may be boundnon-covalently to streptavidin covalently attached to the solid phasesupport. Alternatively, an amino group (—NH₂) may be chemicallyincorporated into one of the PCR primer strands and used to covalentlylink the DNA template to a solid phase support using standard chemistry,such as reactions with N-hydroxysuccinimide activated agarose surfaces.

In another embodiment, the 5′ ends of the sequencing oligonucleotideprimer may be modified with biotin, for non-covalent capture to astreptavidin-treated support, or with an amino group for chemicallinkage to a solid support; the template strands are then captured bythe non-covalent binding attraction between the immobilized primer basesequence and the complementary sequence on the template strands. Methodsfor immobilizing DNA on a solid phase support are well known to those ofskill in the art and will vary depending on the solid phase supportchosen.

In the reactive sequencing method of the present invention, DNApolymerase is presented sequentially with each of the 4 dNTPs. In themajority of the reaction cycles, only incorrect dNTPs will be present,thereby increasing the likelihood of misincorporation of incorrectnucleotides into the extending DNA primer/template system.

Accordingly, the present invention further provides methods foroptimizing the reactive sequencing reaction to achieve rapid andcomplete incorporation of the correct nucleotide into the DNAprimer/template system, while limiting the misincorporation of incorrectnucleotides. For example, dNTP concentrations may be lowered to reducemisincorporation of incorrect nucleotides into the DNA primer. K_(m)values for incorrect dNTPs can be as much as 1000-fold higher than forcorrect nucleotides, indicating that a reduction in dNTP concentrationscan reduce the rate of misincorporation of nucleotides. Thus, in apreferred embodiment of the invention the concentration of dNTPs in thesequencing reactions are approximately 5-20 μM. At this concentration,incorporation rates are as close to the maximum rate of 400nucleotides/s for T4 DNA polymerase as possible.

In addition, relatively short reaction times can be used to reduce theprobability of misincorporation. For an incorporation rate approachingthe maximum rate of ˜400 nucleotides/s, a reaction time of approximately25 milliseconds (ms) will be sufficient to ensure extension of 99.99% ofprimer strands.

In a specific embodiment of the invention, DNA polymerases lacking 3′ to5′ exonuclease activity may be used for reactive sequencing to limitexonucleolytic degradation of primers that would occur in the absence ofcorrect dNTPs. In the presence of all four dNTPs, misincorporationfrequencies by DNA polymerases possessing exonucleolytic proofreadingactivity are as low as one error in 106 to 108 nucleotides incorporatedas discussed in Echols and Goodman (1991, Annu. Rev. Biochem 60;477-511); and Goodman, et al. (1993, Crit. Rev. Biochem. Molec. Biol.28:83-126); and Loeb and Kunkel (1982, Annu. Rev. Biochem. 52:429-457).In the absence of proofreading, DNA polymerase error rates are typicallyon the order of 1 in 10⁴ to 1 in 10⁶. Although exonuclease activityincreases the fidelity of a DNA polymerase, the use of DNA polymeraseshaving proofreading activity can pose technical difficulties for thereactive sequencing method of the present invention. Not only will theexonuclease remove any misincorporated nucleotides, but also, in theabsence of a correct dNTP complementary to the next template base, theexonuclease will remove correctly-paired nucleotides successively untila point on the template sequence is reached where the base iscomplementary to the dNTP in the reaction cell. At this point, an idlingreaction is established where the polymerase repeatedly incorporates thecorrect dNMP and then removes it. Only when a correct dNTP is presentwill the rate of polymerase activity exceed the exonuclease rate so thatan idling reaction is established that maintains the incorporation ofthat correct nucleotide at the 3′ end of the primer.

A number of T4 DNA polymerase mutants containing specific amino acidsubstitutions possess reduced exonuclease activity levels up to10,000-fold less than the wild-type enzyme. For example, Reha-Krantz andNonay (1993, J. Biol. Chem. 268:27100-17108) report that when Asp 112was replaced with Ala and Glu 114 was replaced with Ala (D112A/E114A) inT4 polymerase, these two amino acid substitutions reduced theexonuclease activity on double stranded DNA by a factor of about 300relative to the wild type enzyme. Such mutants may be advantageouslyused in the practice of the invention for incorporation of nucleotidesinto the DNA primer/template system.

In yet another embodiment of the invention, DNA polymerases which aremore accurate than wild type polymerases at incorporating the correctnucleotide into a DNA primer/template may be used. For example, in a(D112A/E114A) mutant T4 polymerase with a third mutation where Ile 417is replaced by Val (1417V/D112A/E114A), the 1417V mutation results in anantimutator phenotype for the polymerase (Reha-Krantz and Nonay, 1994,J. Biol. Chem. 269:5635-5643; Stocki et al., 1995, Mol. Biol.254:15-28). This antimutator phenotype arises because the polymerasetends to move the primer ends from the polymerase site to theexonuclease site more frequently and thus proof read more frequentlythan the wild type polymerase, and thus increases the accuracy ofsynthesis.

In yet another embodiment of the invention, polymerase mutants that arecapable of more efficiently incorporating fluorescent-labelednucleotides into the template DNA system molecule may be used in thepractice of the invention. The efficiency of incorporation offluorescent-labeled nucleotides may be reduced due to the presence ofbulky fluorophore labels that may inhibit dNTP interaction at the activesite of the polymerase. Polymerase mutants that may be advantageouslyused for incorporation of fluorescent-labeled dNTPs into DNA include butare not limited to those described in U.S. application Ser. No.08/632,742 filed Apr. 16, 1996 which is incorporated by referenceherein.

In a preferred embodiment of the invention, the reactive sequencingmethod utilizes a two cycle system. An exonuclease-deficient polymeraseis used in the first cycle and a mixture of exonuclease-deficient andexonuclease-proficient enzymes are used in the second cycle. In thefirst cycle, the primer/template system together with anexonuclease-deficient polymerase will be presented sequentially witheach of the four possible nucleotides. Reaction time and conditions willbe such that a sufficient fraction of primers are extended to allow fordetection and quantification of nucleotide incorporation, ˜98%, foraccurate quantification of multiple single-base repeats. In the secondcycle, after identification of the correct nucleotide, a mixture ofexonuclease proficient and deficient polymerases, or a polymerasecontaining both types of activity will be added in a second cycletogether with the correct dNTP identified in the first cycle to completeand proofread the primer extension. In this way, anexonuclease-proficient polymerase is only present in the reaction cellwhen the correct dNTP is present, so that exonucleolytic degradation ofcorrectly extended strands does not occur, while degradation and correctre-extension of previously incorrectly extended strands does occur, thusachieving extremely accurate strand extension.

The detection of a DNA polymerase mediated extension reaction can beaccomplished in a number of ways. For example, the heat generated by theextension reaction can be measured using a variety of differenttechniques such as those employing thermopile, thermistor and refractiveindex measurements.

In an embodiment of the invention, the heat generated by a DNApolymerase mediated extension reaction can be measured. For example, ina reaction cell volume of 100 micrometers³ containing 1 μg of water asthe sole thermal mass and 2×10¹¹ DNA template molecules (300 fmol)tethered within the cell, the temperature of the water increases by1×10³° C. for a polymerase reaction which extends the primer by a singlenucleoside monophosphate. This calculation is based on the experimentaldetermination that a one base pair extension in a DNA chain is anexothermic reaction and the enthalpy change associated with thisreaction is 3.5 kcal/mole of base. Thus extension of 300 fmol of primerstrands by a single base produces 300 fmol×3.5 kcal/mol or 1×10⁻⁹ cal ofheat. This is sufficient to raise the temperature of 1 pg of water by1×10⁻³° C. Such a temperature change can be readily detectable usingthermistors (sensitivity <10° C.), thermopiles (sensitivity <10⁻⁵° C.);and refractive index measurements (sensitivity <10° C.).

In a specific embodiment of the invention, thermopiles may be used todetect temperature changes. Such thermopiles are known to have a highsensitivity to temperature and can make measurements in the tens ofmicro-degree range in several second time constants. Thermopiles may befabricated by constructing serial sets of junctions of two dissimilarmetals and physically arranging the junctions so that alternatingjunctions are separated in space. One set of junctions is maintained ata constant reference temperature, while the alternate set of junctionsis located in the region whose temperature is to be sensed. Atemperature difference between the two sets of junctions produces apotential difference across the junction set which is proportional tothe temperature difference, to the thermoelectric coefficient of thejunction and to the number of junctions. For optimum response,bimetallic pairs with a large thermoelectric coefficient are desirable,such as bismuth and antimony. Thermopiles may be fabricated using thinfilm deposition techniques in which evaporated metal vapor is depositedonto insulating substrates through specially fabricated masks.Thermopiles that may be used in the practice of the invention includethermopiles such as those described in U.S. Pat. No. 4,935,345, which isincorporated by reference herein.

In a specific embodiment of the invention, miniature thin filmthermopiles produced by metal evaporation techniques, such as thosedescribed in U.S. Pat. No. 4,935,345 incorporated herein by reference,may be used to detect the enthalpy changes. Such devices have been madeby vacuum evaporation through masks of about 10 mm square. Using methodsof photolithography, sputter etching and reverse lift-off techniques,devices as small as 2 mm square may be constructed without the aid ofmodern microlithographic techniques. These devices contain 150thermoelectric junctions and employ 12 micron line widths and canmeasure the exothermic heat of reaction of enzyme-catalyzed reactions inflow streams where the enzyme is preferably immobilized on the surfaceof the thermopile.

To incorporate thermopile detection technology into a reactivesequencing device, thin-film bismuth-antimony thermopiles 2, as shown inFIG. 1, may be fabricated by successive electron-beam evaporation ofbismuth and antimony metals through two differentphotolithographically-generated masks in order to produce a zigzag arrayof alternating thin bismuth and antimony wires which are connected toform two sets of bismuth-antimony thermocouple junctions. Modernmicrolithographic techniques will allow fabrication of devices at leastone order of magnitude smaller than those previously made, i.e., withline widths as small as 1 ÿm and overall dimensions on the order of 100μm². One set of junctions 4 (the sensor junctions) is located within thereaction cell 6, i.e., deposited on a wall of the reaction cell, whilethe second reference set of junctions 8 is located outside the cell at areference point whose temperature is kept constant. Any difference intemperature between the sensor junctions and the reference junctionsresults in an electric potential being generated across the device,which can be measured by a high-resolution digital voltmeter 10connected to measurement points 12 at either end of the device. It isnot necessary that the temperature of the reaction cell and thereference junctions be the same in the absence of a polymerase reactionevent, only that a change in the temperature of the sensor junctions dueto a polymerase reaction event be detectable as a change in the voltagegenerated across the thermopile.

In addition to thermopiles, as shown in FIG. 2, a thermistor 14 may alsobe used to detect temperature changes in the reaction cell 6 resultingfrom DNA polymerase mediated incorporation of dNMPs into the DNA primerstrand. Thermistors are semiconductors composed of a sintered mixture ofmetallic oxides such as manganese, nickel, and cobalt oxides. Thismaterial has a large temperature coefficient of resistance, typically 4%per ° C., and so can sense extremely small temperature changes when theresistance is monitored with a stable, high-resolutionresistance-measuring device such as a digital voltmeter, e.g., KeithleyInstruments Model 2002. A thermistor 14, such as that depicted in FIG.2, may be fabricated in the reactive sequencing reaction cell by sputterdepositing a thin film of the active thermistor material onto thesurface of the reaction cell from a single target consisting of hotpressed nickel, cobalt and manganese oxides. Metal interconnections 16which extend out beyond the wall of the reaction cell may also befabricated in a separate step so that the resistance of the thermistormay be measured using an external measuring device 18.

Temperature changes may also be sensed using a refractive indexmeasurement technique. For example, techniques such as those describedin Bornhop (1995, Applied Optics 34:3234-323) and U.S. Pat. No.5,325,170, may be used to detect refractive index changes for liquids incapillaries. In such a technique, a low-power He—Ne laser is aimedoff-center at a right angle to a capillary and undergoes multipleinternal reflection. Part of the beam travels through the liquid whilethe remainder reflects only off the external capillary wall. The twobeams undergo different phase shifts depending on the refractive indexdifference between the liquid and capillary. The result is aninterference pattern, with the fringe position extremely sensitive totemperature-induced refractive index changes.

In a further embodiment of the invention, the thermal response of thesystem may be increased by the presence of inorganic pyrophosphataseenzyme which is contacted with the template system along with the dNTPsolution. Additionally, heat is released as the pyrophosphate releasedfrom the dNTPs upon incorporation into the template system is hydrolyzedby inorganic pyrophosphatase enzyme.

In another embodiment, the pyrophosphate released upon incorporation ofdNTP's may be removed from the template system and hydrolyzed, and theresultant heat detected, using thermopile, thermistor or refractiveindex methods, in a separate reaction cell downstream. In this reactioncell, inorganic pyrophosphatase enzyme may be mixed in solution with thedNTP removed from the DNA template system, or alternatively theinorganic pyrophosphatase enzyme may be covalently tethered to the wallof the reaction cell.

Alternatively, the polymerase-catalyzed incorporation of a nucleotidebase can be detected using fluorescence and chemiluminescence detectionschemes. The DNA polymerase mediated extension is detected when afluorescent or chemiluminescent signal is generated upon incorporationof a fluorescently or chemiluminescently labeled dNMP into the extendingDNA primer strand. Such tags are attached to the nucleotide in such away as to not interfere with the action of the polymerase. For example,the tag may be attached to the nucleotide base by a linker armsufficiently long to move the bulky fluorophore away from the activesite of the enzyme.

For use of such detection schemes, nucleotide bases are labeled bycovalently attaching a compound such that a fluorescent orchemiluminescent signal is generated following incorporation of a dNTPinto the extending DNA primer/template. Examples of fluorescentcompounds for labeling dNTPs include but are not limited to fluorescein,rhodamine, and BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). See“Handbook of Molecular Probes and Fluorescent Chemicals”, available fromMolecular Probes, Inc. (Eugene, Oreg.). Examples of chemiluminescencebased compounds that may be used in the sequencing methods of theinvention include but are not limited to luminol and dioxetanones (See,Gundennan and McCapra, “Chemiluminescence in Organic Chemistry”,Springer-Verlag, Berlin Heidleberg, 1987).

Fluorescently or chemiluminescently labeled dNTPs are added individuallyto a DNA template system containing template DNA annealed to the primer,DNA polymerase and the appropriate buffer conditions. After the reactioninterval, the excess dNTP is removed and the system is probed to detectwhether a fluorescent or chemiluminescent tagged nucleotide has beenincorporated into the DNA template. Detection of the incorporatednucleotide can be accomplished using different methods that will dependon the type of tag utilized.

For fluorescently-tagged dNTPs the DNA template system may beilluminated with optical radiation at a wavelength which is stronglyabsorbed by the tag entity. Fluorescence from the tag is detected usingfor example a photodetector together with an optical filter whichexcludes any scattered light at the excitation wavelength.

Since labels on previously incorporated nucleotides would interfere withthe signal generated by the most recently incorporated nucleotide, it isessential that the fluorescent tag be removed at the completion of eachextension reaction. To facilitate removal of a fluorescent tag, the tagmay be attached to the nucleotide via a chemically or photochemicallycleavable linker using methods such as those described by Metzger, M.L., et al. (1994, Nucleic Acids Research 22:4259-4267) and Burgess, K.,et al., (1997, J. Org. Chem. 62:5165-5168) so that the fluorescent tagmay be removed from the DNA template system before a new extensionreaction is carried out.

In a further embodiment utilizing fluorescent detection, the fluorescenttag is attached to the dNTP by a photocleavable or chemically cleavablelinker, and the tag is detached following the extension reaction andremoved from the template system into a detection cell where thepresence, and the amount, of the tag is determined by optical excitationat a suitable wavelength and detection of fluorescence. In thisembodiment, the possibility of fluorescence quenching, due to thepresence of multiple fluorescent tags immediately adjacent to oneanother on a primer strand which has been extended complementary to asingle base repeat region in the template, is minimized, and theaccuracy with which the repeat number can be determined is optimized. Inaddition, excitation of fluorescence in a separate chamber minimizes thepossibility of photolytic damage to the DNA primer/template system. Inan additional embodiment utilizing fluorescent detection, the signalfrom the fluorescent tag can be destroyed using a chemical reactionwhich specifically targets the fluorescent moiety and reacts to form afinal product which is no longer fluorescent. In this embodiment, thefluorescent tag attached to the nucleotide base is destroyed followingextension and detection of the fluorescence signal, without the removalof the tag. In a specific embodiment, fluorophores attached to dNTPbases may be selectively destroyed by reaction with compounds capable ofextracting an electron from the excited state of the fluorescent moietythereby producing a radical ion of the fluorescent moiety which thenreacts to form a final product which is no longer fluorescent. In afurther specific embodiment, the signal from a fluorescent tag isdestroyed by photochemical reaction with the cation of adiphenyliodonium salt following extension and detection of thefluorescence label. The fluorescent tag attached to the incorporatednucleotide base is destroyed, without removal of the tag, by theaddition of a solution of a diphenyliodonium salt to the reaction celland subsequent UV light exposure. The diphenyliodonium salt solution isremoved and the reactive sequencing is continued. This embodiment doesnot require dNTP's with chemically or photochemically cleavable linkers,since the fluorescent tag need not be removed.

In a further embodiment of the technique, the response generated by aDNA polymerase-mediated extension reaction can be amplified. In thisembodiment, the dNTP is chemically modified by the covalent attachmentof a signaling tag through a linker that can be cleaved eitherchemically or photolytically. Following exposure of the dNTP to theprimer/template system and flushing away any unincorporated chemicallymodified dNTP, any signaling tag that has been incorporated is detachedby a chemical or photolytic reaction and flushed out of the reactionchamber to an amplification chamber in which an amplified signal may beproduced and detected.

A variety of methods may be used to produce an amplified signal. In onesuch catalytic function. When the catalytic tag is cleaved and allowedto react with chemical reaction ensue producing many moles of productper mole of catalytic multiplication of reaction enthalpy. Either thereaction product is detected, the color or absorbency, or the amplifiedheat product is detected by a thermal signal that is covalently attachedto the dNTP via a cleavable linker arm of sufficient length that doesnot interfere with the active site of the polymerase enzyme. Followingincorporation that enzyme is detached and transported to a secondreactor volume in which specific substrate, thus an amplified responseis obtained as each enzyme of reaction. For example, the enzyme catalase(CAT) catalyzes the reaction:

CAT

H₂O₂→H₂O₂+½O₂+˜100kJ/mol

If each dNTP is tagged with a catalase molecule which is detached afterdNMP incorporation and allowed to react downstream with hydrogenperoxide, each nucleotide incorporation would generate ˜25 kcal/mol×N ofheat where N is the number of hydrogen peroxide molecules decomposed bythe catalase. The heat of decomposition of hydrogen peroxide is already˜6-8 times greater than for nucleotide incorporation, (i.e. 3.5-4kcal/mol). For decomposition of −150 hydrogen peroxide molecules theamount of heat generated per base incorporation approaches 1000 timesthat of the unamplified reaction. Similarly, enzymes which producecolored products, such as those commonly used in enzyme-linkedimmunosorbent assays (ELISA) could be incorporated as detachable tags.For example the enzyme alkaline phosphatase converts colorlessp-nitrophenyl phosphate to a colored product (p-nitrophenol); the enzymehorseradish peroxidase converts colorless o-phenylenediaminehydrochloride to an orange product. Chemistries for linking theseenzymes to proteins such as antibodies are well-known to those versed inthe art, and could be adapted to link the enzymes to nucleotide basesvia linker arms that maintain the enzymes at a distance from the activesite of the polymerase enzymes.

In a further embodiment, an amplified thermal signal may be producedwhen the signaling tag is an entity which can stimulate an activeresponse in cells which are attached to, or held in the vicinity of, athermal sensor such as a thermopile or thermistor. Pizziconi and Page(1997, Biosensors and Bioelectronics 12:457-466) reported that harvestedand cultured mast cell populations could be activated by calciumionophore to undergo exocytosis to release histamine, up to 10-30 pg(100-300 fmol) per cell. The multiple cell reactions leading toexocytosis are themselves exothermic. This process is further amplifiedusing the enzymes diamine oxidase to oxidize the histamine to hydrogenperoxide and imidazoleacetaldehyde, and catalase to disproportionate thehydrogen peroxide. Two reactions together liberate over 100 kJ of heatper mole of histamine. For example, a calcium ionophore is covalentlyattached to the dNTP base via a linker arm which distances the linkedcalcium ionophore from the active site of the polymerase enzyme and ischemically or photochemically cleavable. Following the DNA polymerasecatalyzed incorporation step, and flushing away unincorporatednucleotides any calcium ionophore remaining bound to an incorporatednucleotide may be cleaved and flushed downstream to a detection chambercontaining a mast cell-based sensor such as described by Pizziconi andPage (1997, Biosensors and Bioelectronics 12:457-466). The calciumionophore would bind to receptors on the mast cells stimulatinghistamine release with the accompanying generation of heat. The heatproduction could be further amplified by introducing the enzymes diamineoxidase to oxidize the histamine to hydrogen peroxide andimidazoleacetaldehyde, and catalase to disproportionate the hydrogenperoxide. Thus a significantly amplified heat signal would be producedwhich could readily be detected by a thermopile or thermistor sensorwithin, or in contact with, the reaction chamber.

In a further embodiment utilizing chemiluminescent detection, thechemiluminescent tag is attached to the dNTP by a photocleavable orchemically cleavable linker. The tag is detached following the extensionreaction and removed from the template system into a detection cellwhere the presence, and the amount, of the tag is determined by anappropriate chemical reaction and sensitive optical detection of thelight produced. In this embodiment, the possibility of a non-linearoptical response due to the presence of multiple chemiluminescent tagsimmediately adjacent to one another on a primer strand which has beenextended complementary to a single base repeat region in the template,is minimized, and the accuracy with which the repeat number can bedetermined is optimized. In addition, generation of chemiluminescence ina separate chamber minimizes chemical damage to the DNA primer/templatesystem, and allows detection under harsh chemical conditions whichotherwise would chemically damage the DNA primer/template. In this way,chemiluminescent tags can be chosen to optimize chemiluminescencereaction speed, or compatibility of the tagged dNTP with the polymeraseenzyme, without regard to the compatibility of the chemiluminescencereaction conditions with the DNA primer/template.

In a further embodiment of the invention, the concentration of the dNTPsolution removed from the template system following each extensionreaction can be measured by detecting a change in UV absorption due to achange in the concentration of dNTPs, or a change in fluorescenceresponse of fluorescently-tagged dNTPs. The incorporation of nucleotidesinto the extended template would result in a decreased concentration ofnucleotides removed from the template system. Such a change could bedetected by measuring the UV absorption of the buffer removed from thetemplate system following each extension cycle.

In a further embodiment of the invention, extension of the primer strandmay be sensed by a device capable of sensing fluorescence from, orresolving an image of, a single DNA molecule. Devices capable of sensingfluorescence from a single molecule include the confocal microscope andthe near-field optical microscope. Devices capable of resolving an imageof a single molecule include the scanning tunneling microscope (STM) andthe atomic force microscope (AFM).

In this embodiment of the invention, a single DNA template molecule withattached primer is immobilized on a surface and viewed with an opticalmicroscope or an STM or AFM before and after exposure to buffer solutioncontaining a single type of dNTP, together with polymerase enzyme andother necessary electrolytes. When an optical microscope is used, thesingle molecule is exposed serially to fluorescently-tagged dNTPsolutions and as before incorporation is sensed by detecting thefluorescent tag after excess unreacted dNTP is removed. Again as before,the incorporated fluorescent tag must be cleaved and discarded before asubsequent tag can be detected. Using the STM or AFM, the change inlength of the primer strand is imaged to detect incorporation of thedNTP. Alternatively the dNTP may be tagged with a physically bulkymolecule, more readily visible in the STM or AFM, and this bulky tag isremoved and discarded before each fresh incorporation reaction.

When sequencing a single molecular template in this way, the possibilityof incomplete reaction producing erroneous signal and out-of-phasestrand extension, does not exist and the consequent limitations on readlength do not apply. For a single molecular template, reaction eitheroccurs or it does not, and if it does not, then extension either ceasesand is known to cease, or correct extension occurs in a subsequent cyclewith the correct dNTP. In the event that an incorrect nucleotide isincorporated, which has the same probability as more the multiple strandprocesses discussed earlier, for example 1 in 1,000, an error isrecorded in the sequence, but this error does not propagate or affectsubsequent readout and so the read length is not limited by incorrectincorporation.

Detection and Compensation for DNA Polymerase Errors

In the reactive sequencing process, extension failures will typicallyarise due to the kinetics of the extension reaction and limitations onthe amount of time allotted for each extension trial with the singledeoxynucleotide triphosphates (dNTP's). When reaction is terminated byflushing away the dNTP supply, some small fraction of the primer strandsmay remain unextended. These strands on subsequent dNTP reaction cycleswill continue to extend but will be out of phase with the majoritystrands, giving rise to small out-of-phase signals (i.e. signaling apositive incorporation for an added dNTP which is incorrect forextension of the majority strands). Because extension failure can occur,statistically, on any extension event, these out-of-phase signals willincrease as the population of strands with extension failures grows.Ultimately the out-of-phase signal becomes comparable in amplitude withthe signal due to correct extension of the majority strands and thesequence may be unreadable. The length by which the primer has beenextended when the sequence becomes unreadable is known as the sequencingread length.

The present invention relates to a method that can extend the sequencingread length in two ways, first, by discriminating between the in-phaseand out-of-phase signals, and second by calculating where, and how, adNTP probe sequence can be altered so as selectively to extend theout-of-phase strands to bring them back into phase with the majoritystrands.

Specifically, a method is provided for discriminating between thein-phase and out-of-phase sequencing signals comprising: (i) detectingand measuring error signals thereby determining the size of the trailingstrand population; (ii) between the 3′ terminus of the trailing strandprimers and the 3′ terminus of the leading strand primers; (iii)simulating the occurrence of an extension failure at a point upstreamfrom the 3′ terminus of the leading strands thereby predicting at eachextension step the exact point in the sequence previously traversed bythe leading strands to which the 3′ termini of the trailing strands havebeen extended; (iv) predicting for each dNTP introduced the signal to beexpected from correct extension of the trailing strands; and (v)subtracting the predicted signal from the measured signal to yield asignal due only to correct extension of the leading strand population.

“Upstream” refers to the known sequence of bases correctly incorporatedonto the primer strands. “Downstream” refers to the sequence beyond the3′ terminus. Thus for the leading strand population the downstreamsequence is unknown but is predetermined by the sequence of the templatestrand that has not yet been read; for the trailing strand population,the downstream sequence is known for the gap between the 3′ termini ofthe trailing and leading strands.

The gap between the leading and trailing primer strands may be 1, 2 or 3bases (where a single base repeat of any length, e.g. AAAA, is countedas a single base because the entire repeat will be traversed in a singlereaction cycle if the correct dNTP is introduced), but can never exceed3 bases nor shrink spontaneously to zero if the reaction cycle of thefour dNTP's is unchanged and no other reaction errors occur, for examplea second extension failure on the same primer strand. If the reactioncycle of the four dNTP's is unchanged, it may readily be understood thata primer strand which has failed to extend when the correct dNTP, forexample dATP, is in the reaction chamber cannot trail the leading(majority) strands (which did extend) by more than 3 bases, because thefourth base in the dNTP reaction cycle will always once again be thecorrect base (dATP) for the strand which failed to extend previously.Similarly, a trailing strand resulting from an extension failure cannever re-synchronize with the leading strands if extension subsequentlyproceeds correctly, because the leading strands will always haveextended by at least one more nucleotide—G, T, or C in the examplediscussion of an A extension failure—before the trailing strand can addthe missing A. The effect is that after each complete dNTP cycle thetrailing strands always follow the leading strands by an extensionamount that represents the bases added in one complete dNTP cycle at agiven point in the sequence. A further consequence is that all trailingstrands that have undergone a single failure are in phase with eachother regardless of the point at which the extension failure occurred.

The methods described herein may be utilized to significantly extend theread length that can be achieved by the technique of reactive sequencingby providing a high level of immunity to erroneous signals arising fromextension failure. In a preferred embodiment of the invention, thediscrimination method of the invention is computer based.

First, determination of the readout signals allows real-timediscrimination between the signals due to correct extension of theleading strand population and error signals arising from extension ofthe population of trailing strands resulting from extension failure.Using this information, accurate sequence readout can be obtainedsignificantly beyond the point at which the trailing strand signalswould begin to mask the correct leading strand signals. In fact, becausethe trailing strand signals can always be distinguished from the leadingstrand signals, it is possible to allow the trailing strand populationto continue to grow, at the expense of the leading strands, to the pointwhere the sequence is read from the signals generated on the trailingstrand population, and the leading strand signals are treated as errorsignals to be corrected for. Ultimately, as the probability that aprimer strand will have undergone at least one extension failureapproaches unity, the signals from the leading strand population willdisappear. Correspondingly the probability will increase that a trailingstrand will undergo a second extension failure; the signals from thissecond population of double failure strands can be monitored and thesingle failure strand signals corrected in just the same way as the zerofailure strand signals were corrected for signals due to single failurestrands.

Second, because knowledge of the leading strand sequence permits one toknow the point to which the trailing strands have advanced, bysimulating the effect of an extension failure on that known sequence ina computer, and also to know the sequence in the 1, 2 or 3 base gapbetween these strands and the leading strands, then for a given templatesequence the dNTP probe cycle can be altered at any point to selectivelyextend the trailing strands while not extending the leading strands,thereby resynchronizing the populations. Alternatively the gap betweenleading and trailing strands can be simulated in the computer and thegap can be eliminated by reversing the dNTP cycle whenever the gapshrinks to a single base. These processes are referred to as “healing.”If a large number of different sequences are being read in parallel withthe same dNTP reagents, an altered dNTP probe cycle that is correct forhealing extension failure strands on a given sequence may not be correctfor healing other sequences. However, with a large enough number ofparallel sequence readouts, roughly one-third of the sequences will havetrailing strands with a 1-base gap at any point, and so reversal of thedNTP probe cycle at arbitrary intervals will heal roughly one-third ofthe readouts with extension failure gaps. Repeated arbitrary reversal ofthe dNTP probe cycle eventually heals roughly two-thirds of all thereadouts. The overall effect of these error correction and errorelimination processes is to reduce, or eliminate any limitation on readlength arising from extension failure.

The ability to overcome the read length limitations imposed by extensionfailure provides significant additional flexibility in experimentaldesign. For example, it may be that read length is not limited byextension failure, but rather by misincorporation of incorrectnucleotides, which shuts down extension on the affected strands andsteadily reduces the signal, ultimately to the point where it is notdetectable with the desired accuracy. In this case, the ability toeliminate the effects of extension failure allows the experimenter greatflexibility to alter the reaction conditions in such a way thatmisincorporation is minimized, at the expense of an increased incidenceof extension failure. Misincorporation frequency depends in part on theconcentration of the probing dNTP's and the reaction time allowed.Longer reaction times, or higher dNTP concentrations result in anincreased probability of misincorporation, but a reduced incidence ofextension failure. Therefore, if a higher level of extension failure canbe tolerated due to, for example, the computer-aided signaldiscrimination and dNTP cycle-reversal healing methods, then reactiontimes and/or dNTP reagent concentrations can be reduced to minimizemisincorporation, with the resulting increase in extension failure beingcountered by the computer-aided signal discrimination and/or dNTPcycle-reversal healing techniques described above.

If the deoxyribonucleotides used for the polymerase reaction are impurea small fraction of strands will extend when the main nucleotide isincorrect and produce a population of leading, rather than trailing,error strands. As with the trailing strands, the leading strandpopulation is never more than three bases, nor less than one base, aheadof the main population, unless a second error occurs on the same strand,and also, regardless of where an incorrect extension by an impurity dNTPoccurs, the leading strands are all in phase with each other. A givenbase site can be probed either 1, 2 or 3 times with an incorrect dNTPbefore it must be extended by the correct dNTP, so on the average twice.If each of the incorrect dNTP's is assumed to carry the same percentageof dNTP impurity, then the probability of incorrect extension by, e.g.99% pure dNTP containing the correct complementary base as an impurityis 1%+3 (only ⅓ of the impurity will be the correct complementarybase)×2 (average 2 incorrect trials between each correct extension),that is, 0.67%.

As with trailing strands, the leading strand population can produceout-of-phase extension signals that complicate the readout of themajority strand sequence, as shown in FIG. 15. Because the sequencedownstream of the 3′ terminus of the majority strands is not known atthe time of extension of those strands, the signal due to leading strandextension can not immediately be corrected for, nor can an altered dNTPcycle be calculated which would automatically heal the gap betweenmajority and leading strands for a given template sequence. Howeversimilar methods can be used to ameliorate the effects of a leadingstrand population. First, as with trailing strands, reversal of the dNTPprobe cycle automatically heals the gap between leading and majoritystrand populations whenever the gap shrinks to a single base. Therefore,arbitrary reversal of the dNTP probe cycle has a ⅓ probability ofhealing the gap for a given sequence, or will heal ⅓ of the sequences ina large population of sequences probed in parallel. Continued arbitraryreversal eventually heals roughly two-thirds of such gaps. Second,although the sequence downstream of the 3′ terminus of the majoritystrands is not immediately known, information about this sequencebecomes available as soon as the majority strands traverse the gapregion. Therefore, for each extension of the majority strands it ispossible, ideally using a computer simulation, to calculate when theleading strand population would have traversed that base and thus thesignal by which a prior extension of the majority strands would havebeen contaminated. In this way the majority strand extension signals canretrospectively be corrected for leading strand signals.

There are important aspects to leading strand creation that reduce thefrequency of occurrence of leading strand events. First, if theconcentration of impurity dNTP's is sufficiently low, a leading strandpopulation cannot be created by impurity extension of the first base ofa repeat. This is because the probability of incorrect incorporation oftwo impurity bases on the same strand in the same reaction cycle is thesquare of the probability for a single incorporation, and thereforevanishingly small for small impurity levels. Therefore, whenever thecorrect dNTP for extension of the repeat length is supplied, all strandswill be extended to completion when the correct nucleotide is supplied,regardless of whether some fraction of the strands were alreadypartially extended by one base of the repeat. Second, not all incorrectextensions result in a permanent phase difference. For a permanent phasedifference to result, a second extension (by a correct base) must occuron the leading strand before the main strands extend to catch up to theleading strand. Labeling the next four sites along the templatesequence: 1, 2, 3, 4, then, by definition, if a leading strand iscreated by incorporation of an impurity base on site 1 while themajority of the strands do not extend, the main nucleotide supplied isincorrect for extension at site 1. If the main nucleotide supplied iscorrect for extension at site 2, a 2-base lead is created. There is 1chance in 4 that the reaction chamber contains the correct nucleotidefor site 2, so the probability of creating a 2-base extension in asingle step (with an impurity extension followed by a correct extension)is 114 the probability of the impurity extension alone. For the 0.67%impurity extension probability cited above, this means a 0.16%probability of creating a 2-base extension in a single cycle.

However, if the main nucleotide supplied is incorrect for furtherextension at site 2, and, by definition incorrect for extension at site1, then for the lead to become fixed, the correct nucleotide for site 2must be supplied before the correct nucleotide to extend at site 1. Theprobability that site 2 will extend before site 1 is therefore 50%; fora 0.67% impurity extension probability, the probability that thiscreates a fixed lead due to a second extension by a correct nucleotideis 0.33%. Overall, a 1% impurity level results in ˜0.5% probability ofcreating a leading strand in any given reaction trial.

Preparation of specific embodiments in accordance with the presentinvention will now be described in further detail. These examples areintended to be illustrative and the invention is not limited to thespecific materials and methods set forth in these embodiments.

Example 1

A microcalorimetic experiment was performed which demonstrates for thefirst time the successful thermal detection of a DNA polymerasereaction. The results are shown in FIG. 3. Approximately 20 units of 17Sequenase was injected into a 3 mL reaction volume containingapproximately 20 nmol of DNA template and complementary primer, and anexcess of dNTPs. The primer was extended by 52-base pairs, the expectedlength given the size of the template. Using a commercialmicrocalorimeter (TAM Model 2273; Thermometrics, Sweden) a reactionenthalpy of 3.5-4 kcal per mole of base was measured (FIG. 3). Thismeasurement is well within the value required for thermal detection ofDNA polymerase activity. This measurement also demonstrates thesensitivity of thermopile detection as the maximum temperature rise inthe reaction cell was 1×10⁻³ C. The lower trace seen in FIG. 3 is from areference cell showing the injection artifact for an enzyme-freeinjection into buffer containing no template system.

Example 2

To illustrate the utility of mutant T4 polymerases, two primer extensionassays were performed with two different mutant T4 polymerases, both ofwhich are exonuclease deficient. In one mutant, Asp112 is replaced withAla and G1u114 is replaced with Ala (D112A/E114A). The exonucleaseactivity of this mutant on double-stranded DNA is reduced by a factor ofabout 300 relative to the wild type enzyme as described by Reha-Krantzand Nonay (1993, J. Biol. Chem. 268:27100-27108). In a second polymerasemutant, in addition to the DI12A/E114A amino acid substitutions, a thirdsubstitution replace Ile417 with Val (1417V/D112A/E114A). The I417Vmutation increases the accuracy of synthesis by this polymerase (Stocki,S. A. and Reha-Krantz, L. J, 1995, J. Mol. Biol. 245:15-28; Reha-Krantz,L. J. and Nonay, R. L., 1994, J. Biol. Chem. 269:5635-5643).

Two separate primer extension reactions were carried out using each ofthe polymerase mutants. In the first, only a single correct nucleotide,dGTP, corresponding to a template C was added; The next unpairedtemplate site is a G so that misincorporation would result in formationof a G-G mispair. A G-G mispair tends to be among the most difficultmispairs for polymerases to make. In the second primer extensionreaction, two nucleotides, dGTP and dCTP, complementary to the firstthree unpaired template sites were added. Following correctincorporation of dGMP and dCMP, the next available template site is a T.Formation of C-T mispairs tend to be very difficult while G-T mispairstend to be the most frequent mispairs made by polymerases.

Time courses for primer extension reactions by both mutant T4polymerases are shown in FIG. 4. Low concentrations of T4 polymeraserelative to primer/template (p/t) were used so that incorporationreactions could be measured on convenient time scales (60 min). By 64minutes 98% of the primers were extended. In reactions containing onlydGTP, both polymerases nearly completely extended primer ends by dGMPwithout any detectable incorporation of dGMP opposite G. In reactionscontaining both dGMP and dCMP, both polymerases nearly completelyextended primer ends by addition of one dGMP and two dCMP's. A smallpercentage (≈1%) of misincorporation was detectable in the reactioncatalyzed by the D112A/E114Amutant. Significantly, no detectablemisincorporation was seen in the reaction catalyzed by theI417V/D112A/E114A mutant

Example 3

In accordance with the invention a fluorescent tag may be attached tothe nucleotide base at a site other than the 3′ position of the sugarmoiety. Chemistries for such tags which do not interfere with theactivity of the DNA polymerase have been developed as described byGoodwin et al. (1995, Experimental Technique of Physics 41:279-294).Generally the tag is attached to the base by a linker arm of sufficientlength to move the bulky tag out of the active site of the enzyme duringincorporation.

As illustrated in FIG. 5, a nucleotide can be connected to a fluorophoreby a photocleavable linker, e.g., a benzoin ester. After the tagged dNMPis incorporated onto the 3′ end of the DNA primer strand, the DNAtemplate system is illuminated by light at a wave length correspondingto the absorption maximum of the fluorophore and the presence of thefluorophore is signaled by detection of fluorescence at the emissionmaximum of the fluorophore. Following detection of the fluorophore, thelinker may be photocleaved to produce compound 2; the result is anelongated DNA molecule with a modified but non-fluorescent nucleotideattached. Many fluorophores, including for example, a dansyl group oracridine, etc., will be employed in the methodology illustrated by FIG.5.

Alternatively, the DNA template system is not illuminated to stimulatefluorescence. Instead, the photocleavage reaction is carried out toproduce compound 2 releasing the fluorophore, which is removed from thetemplate system into a separate detection chamber. There the presence ofthe fluorophore is detected as before, by illumination at the absorptionmaximum of the fluorophore and detection of emission near the emissionmaximum of the fluorophore.

Example 4

In a specific embodiment of the invention, a linked system consisting ofa chemiluminescently tagged dNTP can consist of a cherniluminescentgroup (the dioxetane portion of compound 4), a chemically cleavablelinker (the silyl ether), and an optional photocleavable group (thebenzoin ester) as depicted in FIG. 6. The cleavage of the silyl ether bya fluoride ion produces detectable chemiluminescence as described inSchaap, et al. (1991, “Chemical and Enzymatic Triggering of1,2-dioxetanes: Structural Effects on Chemiluminescence Efficiency” inBioluminescence & Chemiluminescence, Stanley, P. E. and Knicha, L. J.(Eds), Wiley, N.Y. 1991, pp. 103-106). In addition, the benzoin esterthat links the nucleoside triphosphate to the silyl linker isphotocleavable as set forth in Rock and Chan (1996, J. Org. Chem. 61:1526-1529); and Felder, et al. (1997, First International ElectronicConference on Synthetic Organic Chemistry, Sep. 1-30). Having both achemiluminescent tag and a photocleavable linker is not alwaysnecessary; the silyl ether can be attached directly to the nucleotidebase and the chemiluminescent tag is destroyed as it is read.

As illustrated in FIG. 6 with respect to compound 3, treatment withfluoride ion liberates the phenolate ion of the adamantyl dioxetane,which is known to chemiluminesce with high efficiency (Bronstein et al.,1991, “Novel Chemiluminescent Adamantyl 1,2-dioxetane EnzymeSubstrates,” in Bioluminescence & Chemiluminescence, Stanley, R E. andKricka, R. J. (eds), Wiley, N.Y. 1991 pp. 73-82). The other product ofthe reaction is compound 4, which is no longer chemiluminescent.Compound 4 upon photolysis at 308-366 nm liberates compound 2.

The synthesis of compound 1 is achieved by attachment of the fluorophoreto the carboxyl group of the benzoin, whose a-keto hydroxyl group isprotected by 9(FMOC), followed by removal of the FMOC protecting groupand coupling to the nucleotide bearing an activated carbonic acidderivative at its 3′ end. Compound 4 is prepared via coupling of thevinyl ether form of the adamantyl phenol, tochloro(3-cyanopropyl)dimethylsilane, reduction of the cyano group to theamine, generation of the oxetane, and coupling of this chemiluminescenceprecursor to the nucleotide bearing an activated carbonic acidderivative at its 3′ end.

The chemiluminescent tag can also be attached to the dNTP by a cleavablelinkage and cleaved prior to detection of chemiluminescence. As shown inFIG. 7, the benzoin ester linkage in compound 3 may be cleavedphotolytically to produce the free chemiluminescent compound 5. Reactionof compound 5 with fluoride ion to generate chemiluminescence may thenbe carried out after compound 5 has been flushed away from the DNAtemplate primer in the reaction chamber. As an alternative to photolyticcleavage, the tag may be attached by a chemically cleavable linker whichis cleaved by chemical processing which does not trigger thechemiluminescent reaction.

Example 5

In this example, the nucleotide sequence of a template moleculecomprising a portion of DNA of unknown sequence is determined. The DNAof unknown sequence is cloned into a single stranded vector such as M13.A primer that is complementary to a single stranded region of the vectorimmediately upstream of the foreign DNA is annealed to the vector andused to prime synthesis in reactive sequencing. For the annealingreaction, equal molar ratios of primer and template (calculated based onthe approximation that one base contributes 330 g/mol to the molecularweight of a DNA polymer) is mixed in a buffer consisting of 67 mMTrisHCl pH 8.8, 16.7 mM (NH₄)₂SO₄ and 0.5 mM EDTA. This buffer issuitable both for annealing DNA and subsequent polymerase extensionreactions. Annealing is accomplished by heating the DNA sample in bufferto 80° C. and allowing it to slowly cool to room temperature. Samplesare briefly spun in a microcentrifuge to remove condensation from thelid and walls of the tube. To the DNA is added 0.2 mol equivalents of T4polymerase mutant I417V/D112NE114A and buffer components so that thefinal reaction cell contains 67 mM TrisHCl pH 8.8, 16.7 mM (NH₄)₂SO₄,6.7 mM MgCl₂ and 0.5 mM dithiothreitol. The polymerase is then queriedwith one dNTP at a time at a final concentration of 10 ÿM. Thenucleotide is incubated with polymerase at 37° C. for 10 s.Incorporation of dNTPs may be detected by one of the methods describedabove including measuring fluorescence, chemiluminescence or temperaturechange. The reaction cycle will be repeated with each of the four dNTPsuntil the complete sequence of the DNA molecule has been determined.

Example 6

FIG. 7 illustrates a mechanical fluorescent sequencing method inaccordance with the invention. A DNA template and primer are capturedonto beads 18 using, for example, avidin-biotin or —NH₂/nhydroxysuccinimide chemistry and loaded behind a porous frit or filter20 at the tip of a micropipette 22 or other aspiration device as shownin FIG. 7( a), step 1. Exonuclease deficient polymerase enzyme is addedand the pipette tip is lowered into a small reservoir 24 containing asolution of fluorescently-labeled dNTP. As illustrated in step 2 of FIG.7( a), a small quantity of dNTP solution is aspirated through the filterand allowed to react with the immobilized DNA. The dNTP solution alsocontains approximately 100 nM polymerase enzyme, sufficient to replenishrinsing losses. After reaction, as shown in step 3, the excess dNTPsolution 24 is forced back out through the frit 20 into the dNTPreservoir 24. In step 4 of the process the pipette is moved to areservoir containing buffer solution and several aliquots of buffersolution are aspirated through the frit to rinse excess unbound dNTPfrom the beads. The buffer inside the pipette is then forced out anddiscarded to waste 26. The pipette is moved to a second buffer reservoir(buffer 2), containing the chemicals required to cleave the fluorescenttag from the incorporated dNMP. The reaction is allowed to occur tocleave the tag. As shown in step 5 the bead/buffer slurry with thedetached fluorescent tag in solution is irradiated by a laser or lightsource 28 at a wavelength chosen to excite the fluorescent tag, thefluorescence is detected by fluorescence detector 30 and quantified ifincorporation has occurred.

Subsequent steps depend on the enzyme strategy used. If a single-stagestrategy with an exonuclease-deficient polymerase is used, asillustrated in FIG. 7( b), the solution containing the detachedfluorescent tag is discarded to waste (step 6) which is expelled,followed by a further rinse step with buffer 1 (step 7) which isthereafter discarded (step 8) and the pipette is moved to a secondreservoir containing a different dNTP (step 9) and the process repeatsstarting from step 3, cycling through all four dNTPs.

In a two-stage strategy, after the correct dNTP has been identified andthe repeat length quantified in step 5, the reaction mixture is rinsedas shown in steps 6, 7, and 8 of FIG. 7( b) and the pipette is returnedto a different reservoir containing the same dNTP (e.g., dentil) asshown in step (a) of FIG. 8 to which a quantity ofexonuclease-proficient polymerase has been added and the solution isaspirated for a further stage of reaction which proof-reads the priorextension and correctly completes the extension. This second batch ofdNTP need not be fluorescently tagged, as the identity of the dNTP isknown and no sequence information will be gained in this proof-readingstep. If a tagged dNTP is used, the fluorescent tag is preferablycleaved and discarded as in step 5 of FIG. 7( a) using Buffer 2.Alternatively, the initial incorporation reaction shown in step 2 ofFIG. 7( a) is carried out for long enough, and the initial polymerase isaccurate enough, so that the additional amount of fluorescent tagincorporated with dNTP1 at step a of FIG. 8 is small and does notinterfere with quantification of the subsequent dNTP. Followingproof-reading in step a of FIG. 8, excess dNTP is expelled (step b) andthe reaction mixture is rinsed (steps c, d) with a high-salt buffer todissociate the exo+polymerase from the DNA primer/template. It isimportant not to have exonuclease-proficient enzyme present if the DNAprimer/template is exposed to an incorrect dNTP. The pipette is thenmoved to step e, in which the reservoir contains a different dNTP, andthe process is repeated, again cycling through all four dNTPs.

Example 7

A new process for destruction of a fluorophore signal which involvesreaction of the electronically excited fluorophore with anelectron-abstracting species, such as diphenyliodonium salts, isdescribed.

The reaction of a diphenyliodonium ion with an electronically excitedfluorescein molecule is illustrated in FIG. 10. The diphenyliodonium ionextracts an electron from the excited state of the fluorescein moleculeproducing a radical ion of the fluorescein molecule and a neutraldiphenyliodonium free radical. The diphenyliodonium free radical rapidlydecomposes to iodobenzene and a phenyl radical. The fluorescein radicalion then either reacts with the phenyl radical or undergoes an internalarrangement to produce a final product which is no longer fluorescent.

FIGS. 11 and 12 demonstrate evidence for the specific destruction offluorescein by diphenylionium ion. In FIG. 11, fluorescence spectra arepresented for a mixture of fluorescein and tetramethylrhodamine dyes,before and after addition of a solution of diphenyliodonium chloride. Itis seen that the fluorescence from the fluorescein dye is immediatelyquenched, demonstrating electron abstraction from the excited state ofthe molecule while the fluorescence from the rhodamine is unaffected,apart from a small decrease due to the dilution of the dye solution bythe added diphenyliodonium chloride solution.

Elimination of the fluorescent signal from the fluorescein dye bydiphenyliodonium chloride is not in itself proof that the fluoresceinmolecule has been destroyed, because electron abstraction from theexcited state of fluorescein effectively quenches the fluorescence, andquenching need not result in destruction of the fluorescein molecule.However, FIG. 12 demonstrates that the fluorescein molecule is destroyedby reaction with the diphenyliodonium and not simply quenched. FIG. 12demonstrates the ultraviolet (UV) absorption spectra for a fluoresceinsolution before and after addition of a solution of diphenyliodoniumchloride. Spectrum 1 is the UV absorption spectrum of a pure fluoresceinsolution. Spectrum 2 is the UV absorption of the fluorescein solutionfollowing the addition of a solution containing a molar excess ofdiphenyliodonium (DPI) chloride and exposure to a single flash from axenon camera strobe. The data show that fluorescein is essentiallydestroyed by the photochemical reaction with the DPI ion. FIG. 12provides clear evidence that diphenyliodonium chloride not only quenchesthe fluorescence from the fluorescein dye but destroys the molecule tosuch an extent that it can no longer act as a fluorophore.

An experiment was performed to demonstrate efficient fluorescentdetection and destruction of fluorophore using a template sequence. Thetemplate, synthesized with a alkylamino linker at the 5′ terminus, was:

3′-H₂N—(CH₂)₇-GAC CAT TAT AGG TCT TGT TAG GGA AAG GAA GA-5′  (SEQ ID NO:2)

The trial sequence to be determined is: G GGA AAG GAA GA (SEQ ID NO: 3).

A tetramethyrhodamine-labeled primer sequence was synthesized to becomplementary to the template as follows:

5′-[Rhodamine]-(CH₂)₆-CTG GTA ATA TCC AGA ACA AT-3′  (SEQ ID NO: 4)

The alkyl amino-terminated template molecules were chemically linked toSepharose beads derivatized with N-hydroxysuccinimide and therhodamine-labeled primer was annealed to the template. The beads withattached DNA template and annealed primer were loaded behind a B-100disposable filter in a 5-ml syringe. A volume containing a mixture offluorescein-labeled and unlabelled dCTP in a ratio of 1:2 andexonuclease-deficient polymerase enzyme in a reaction buffer asspecified by the manufacturer was drawn into the syringe. Reaction wasallowed to proceed for 20 minutes, at 35° C. After the reaction, thefluid was forced out of the syringe, retaining the beads with thereacted DNA behind the filter, and three washes with double-distilledwater were performed by drawing water through the filter into thesyringe and expelling it. The beads were resuspended in phosphatebuffer, the filter was removed and the suspension was dispensed into acuvette for fluorescence analysis. Following fluorescence analysis, thebead suspension was loaded back into the syringe which was then fittedwith a filter tip, and the phosphate buffer was dispensed. A solution ofDPI was drawn up into the syringe with a concentration calculated to bein 1:1 molar equivalence to the theoretical amount of DNA template, thefilter was removed and the bead suspension was dispensed into a cuvettefor UV light exposure for 15 minutes. The suspension was recollectedinto a syringe, the filter was reattached, the DPI solution wasexpelled, and the beads were resuspended by drawing up 0.7 mL ofphosphate buffer. After removal of the filter the bead suspension wasdispensed into a clean cuvette for fluorescence analysis to check thecompleteness of destruction of the fluorescein by the reaction with theDPI. A subsequent polymerase reaction was performed using the sameprotocol with labeled dTTP and similarly measured for fluorescence.

FIG. 13 demonstrates the results of the polymerase reactions, withphotochemical destruction of the fluorescein label by DPI following eachnucleotide incorporation reaction. Curve 1 shows rhodamine fluorescencefollowing annealing of the rhodamine labeled primer to the beads,demonstrating covalent attachment of the template strands to the beadsand capture of the rhodamine-labeled primer strands. Curve 2demonstrates detection of fluorescein following polymerase-catalyzedincorporation of three partially fluorescein-labeled dCMPs onto the 3′terminus of the primer strands. Curve 3 shows complete destruction ofthe incorporated fluorescein label by photo-induced reaction withdiphenyliodonium chloride. Loss of rhodamine signal here is attributedto loss of a significant fraction of the beads which stuck to the filterduring washes. Curve 4 shows detection of a new fluorescein labelfollowing photochemical destruction of the fluorescein attached to thedCMP's and subsequent polymerase-catalyzed incorporation of threepartially fluorescein-labeled dTMPs onto the 3′ terminus of the primerstrands.

The following methods were utilized to demonstrate successfuldestruction of a fluorescein-labeled dTMP.

Sepharose beads were purchased from Amersham with surfaces derivatizedwith N-hydroxysuccinimide for reaction with primary amine groups. Thealkyl amino-terminated templates were chemically linked to the Sepharosebeads using the standard procedure recommended by the manufacturer.

The beads with attached template were suspended in 250 mM Tris buffercontaining 250 mM NaCl and 40 nM MgCl₂. The solution containing theprimer strands was added and the mixture heated to 80° C. and cooledover ˜2 hours to anneal the primers to the surface-immobilized DNAtemplate strands.

Fluorescein-labeled dUTP and dCTP were purchased from NEN Life ScienceProducts. Unlabeled dTTP and dCTP were purchased from Amersham.

Prior to any reaction, the annealed primer/template was subjected tofluorescence analysis to ensure that annealing had occurred. Theexcitation wavelength used was 320 nm and fluorescence from fluoresceinand rhodamine was detected at ˜520 nm and ˜580 nm respectively.

Reagent volumes were calculated on the assumption that the DNA templatewas attached to the beads with 100% efficiency.

The 5× reaction buffer contained:

1) 250 mM Tris buffer, pH 7.5 2) 250 mM NaCl 3) 40 mM MgCl₂ 4) 1 mg/mLBSA 5) 25 mM dithiothreitol (DTT) — mixed and brought to volume withdouble-

T4 DNA polymerase was obtained from Worthington Biochemical Corp. Thepolymerase was dissolved in the polymerase buffer according to themanufacturer's protocols.

Fluorescein-labeled and unlabeled dCTP's were mixed in a ratio of 1:2.

The reaction was run in a 5 mL syringe (Becton Dickinson) fitted with aB-10 (Upchurch Scientific). This limits the reaction volume to 5 mLtotal:

Primer template suspension 0.7 mL T4 DNA Polymerase 1.0 mL FdCTP/dCTP0.040 mL 5X reaction buffer 2.0 mL double-dist. H₂O 1.0 mL

The reaction was allowed to proceed in a 35° C. oven for 20 minutes.

Following reaction, the fluid was forced out of the syringe allowing thefilter to retain the beads with the reacted DNA. Three washes withdouble-distilled water were performed. All waste was collected and savedfor future reuse. The beads were resuspended in 0.7 mL of phosphatebuffer, the filter was removed and the suspension was dispensed into acuvette for fluorescence analysis.

Following fluorescence analysis the bead suspension was collected into a1 mL syringe (Becton Dickinson) which was then fitted with a filter tip.The phosphate buffer was dispensed and the waste collected. A solutionof diphenyliodonium chloride (DPI) was drawn up with a concentrationcalculated to be in 1:1 molar equivalence to the theoretical amount ofDNA template (i.e. DPI was present in excess of the incorporatedfluorescein-labeled dCTP). The filter was removed and the beadsuspension with added DPI was dispensed into a cuvette and exposed to UVlight for 15 minutes. The suspension was recollected into a syringe, thefilter reattached, the DPI solution was dispensed and the beads wereresuspended in 0.7 mL of phosphate buffer. The bead suspension wasdispensed into a clean cuvette for fluorescence analysis.

It should be noted that a significant fraction of the beads used in thisprocedure appeared to become stuck in the filter on the syringe. Thisresulted in a significant increase in the pressure needed to forcefluids through the filter as it became clogged by the beads, and moreimportantly reduced the amount of DNA available for fluorescentdetection of incorporated nucleotides and reduced the weak rhodaminesignal from the labeled primer to the point where it was no longerdetectable.

Following the successful incorporation reaction with dCTP, a subsequentpolymerase reaction was run to incorporate dTTP. The incorporatedfluorescein-labeled dTMP was detected, but with significantly lowerintensity due to the losses of the beads in the filter in the multipletransfer steps between the reaction syringe and the analysis cuvette.The lowered signal could also result in part from a different labelingefficiency of the dTTP and a different incorporation efficiency for thelabeled nucleotide in the polymerase reaction. Because the rhodaminesignal was no longer detectable following the second incorporationreaction it was not possible to correct for bead losses.

The results are shown in FIG. 13. The data represented by the curveswere obtained sequentially as follows:

Curve 1 shows the rhodamine fluorescence following annealing of therhodamine-labeled primer to the bead-immobilized DNA template.

Curve 2 demonstrates detection of the fluorescein-labeled dCTP followingpolymerase-catalyzed incorporation of three dCMP's onto the 3′ terminusof the primer strands.

Curve 3 demonstrates complete destruction of the incorporatedfluorescein label on the dCMP's by photo-induced reaction withdipenyliodonium chloride. In this instance, the rhodamine label also hasvanished; this is primarily because a significant fraction of the beadswere lost by sticking in the filter used in the reagent flushingoperation. It is possible that the rhodamine also was destroyed by theDPI photochemical reaction.

Curve 4 demonstrates detection of a new fluorescein label followingphotochemical destruction of the fluorescein label on the dCMP's andpolymerase-catalyzed incorporation of three fluorescein-tagged dTMP'sonto the 3′ terminus of the primer strands. The lower signal compared tocurve 2 results mainly from the bead losses in the syringe, but may alsoreflect a lower incorporation efficiency of the dTMP and/or a lowerlabeling efficiency. Because the rhodamine signal from the labeledprimer is no longer detectable, the bead losses cannot be calibrated.

The results shown here demonstrate the concept of reactive sequencing byfluorescent detection of DNA extension followed by photochemicaldestruction of the fluorophore, which allows further extension anddetection of a subsequent added fluorophore. This cycle can be repeateda large number of times if sample losses are avoided. In practicalapplications of this approach, such losses will be avoided by attachingthe primer or template strands to the fixed surface of an array device,for example a microscope slide, and transferring the entire array devicebetween a reaction vessel and the fluorescent reader.

Example 8

Read length is defined as the maximum length of DNA sequence that can beread before uncertainties in the identities of the DNA bases exceed somedefined level. In the reactive sequencing approach, read length islimited by two types of polymerase failures: misincorporation, i.e.,incorrectly incorporating a noncomplementary base, and extensionfailure, i.e., failure to extend some fraction of the DNA primer strandson a given cycle in the presence of the correct complementary base.Example 2 demonstrated that reaction conditions can be optimized suchthat neither type of failure affects more than −1% of the arrayedstrands for any given incorporation reaction. Neither type of failuredirectly produces an error signal in the sequence readout, becauseneither a 1% positive signal, for a misincorporation, nor a 1% decreasein the signal for a correct incorporation, in the case of extensionfailure, will be significant compared to the signals anticipated for acorrect incorporation. However, accumulated failures limit the readlength in a variety of different ways.

For example, misincorporation inhibits any further extension on theaffected strand resulting in a reduction in subsequent signals. It isestimated that the probability of continuing to extend a given strandfollowing a misincorporation is no greater than 0.1%, so that anycontribution to the fluorescent signal resulting from misincorporationfollowed by subsequent extension of the error strand will be negligible.Instead, the accumulation of misincorporations resulting in inhibitionof strand extension ultimately reduces the overall signal amplitude forcorrect base incorporation to a level at which noise signals in thedetection system begin to have a significant probability of producing afalse signal that is read as a true base incorporation.

Extension failures typically arise due to the kinetics of the extensionreaction and limitations on the amount of time allotted for eachextension trial with the single deoxynucleotide triphosphates (dNTP's).When reaction is terminated by flushing away the dNTP supply, a smallfraction of the primer strands may remain unextended. These strands onsubsequent dNTP reaction cycles will continue to extend but will be outof phase with the majority strands, giving rise to small out-of-phasesignals, i.e., signaling a positive incorporation for an added dNTPwhich is incorrect for extension of the majority strands. Becauseextension failure can occur, statistically, on any extension event, theout-of-phase signals will increase as the population of strands withextension failures grows. If reaction conditions are chosen so that thereaction is 99.9% complete on a given reaction cycle, for example, aftera further number, N, of successful extension reactions, the out-of-phasesignal will be approximately (1-0.999^(N)). The number N at which theout-of-phase signal becomes large enough to be incorrectly read as acorrect extension signal is the read length. For example, afterextension by 200 bases with 99.9% completion, the out-of-phase signal isapproximately 18% of the in-phase signal, for a single base extension ineither case. After extension by 400 bases the out-of-phase signal growsto 33%. The point at which the read must terminate is dictated by theability to distinguish the in-phase signals from the out-of-phasesignals.

In what follows, a length of single base repeats, e.g. AAAAA, is treatedas a single base for the purposes of discussing the phase differencebetween strands. If the reaction cycle of the four dNTP's is unchanged,then a primer strand which has failed to extend when the correct dNTP,for example dATP, is in the reaction cell cannot trail the leading,i.e., majority strands, which did extend correctly, by more than 3 basesbecause the fourth base in the dNTP reaction cycle will always onceagain be the correct base (dATP) for the strand which failed to extendpreviously. It is assumed that extension failure is purely statistical,and that any strand which fails to extend has an equal chance ofsubsequent extension when the correct dNTP is supplied, and that thisextension probability is sufficiently high that the chance of repeatedextension failures on the same strand is vanishingly small. For example,if the probability of extension failure on a single strand is 0.1%, theprobability of two extension failures on the same strand is (0.001)² or10⁻⁶. Similarly, the trailing strand can never resynchronize with theleading strands if extension subsequently proceeds correctly, becausethe leading strands will always have extended by at least one morenucleotide—G, T, or C in the example discussion of an A extensionfailure—before the trailing strand can add the missing A. The effect isthat after each complete dNTP cycle the trailing strands always followthe leading strands by an extension amount that represents the basesadded in one complete dNTP cycle at a given point in the sequence. Theseobservations predict that: (i) the gap between the leading and trailingstrands perpetually oscillates between 1 and 3 bases and can neverincrease unless a second extension failure occurs on the same strand;and (ii) the gap between the leading and trailing strands is independentof the position along the trailing strand at which the extension failureoccurs. This gap at any given point in the extension of the leadingstrands is solely a function of the sequence of the leading strandpopulation up to that point and the dNTP probe cycle. In other words, apopulation of trailing strands is produced due to random extensionfailure at different points in the sequence, but these trailing strandsthemselves are all exactly in phase with each other.

Because the result of an extension failure is to produce a trailingstrand population that trails the leading strands perpetually by anamount that oscillates between one and three nucleotides, assuming thata second extension failure does not occur on the trailing strand andthat the probing dNTP cycle remains unchanged, therefore the gap betweenthe leading and trailing strand populations can always be known bytracking the leading strand sequence by, for example, computersimulation and simulating an extension failure event at any point alongthe sequence.

Thus the present invention provides, first, a general method of computertracking of the sequence information which allows the out of-phase errorsignals due to extension of trailing strands to be recognized andsubtracted from the correct signals, and, second, methods of alteringthe probing dNTP cycle to selectively extend the trailing strands sothat they move back into phase with the leading strands, thus completelyeliminating sequence uncertainty due to out of-phase signals arisingfrom the trailing strands that result from extension failure.

The statistics which govern the ability to distinguish an incorrectsignal from out-of-phase strands from a correct signal depend upon thenoise level and statistical variation of the fluorescence signal.Assuming that the signal for a correct 1-base extension has a standarddeviation of ±5%, then statistically 99.75% of the signals will have anamplitude between 0.85 and 1.15 (±3 standard deviations from the averagevalue) when the average value is 1.0 and the standard deviation is 0.05.If the extension signal must be at least 85% of the average singleextension signal to register a correct extension, then statistically acorrect extension will be missed only 0.13% of the time, i.e. thereadout accuracy would be 99.87%. Another 0.13% of the signals for acorrect extension will be greater than 1.15, but the concern is onlywith signals that are lower than average and so are more difficult todistinguish from a growing signal from out-of-phase strands. Thestatistics for errors arising from out-of-phase extension of a trailingstrand are similar. If the standard deviation of the trailing strandsignals is also ±5% of the mean extension signal which will be truewhenever the trailing strand intensity approaches the leading strandintensity, then if the trailing strand intensity does not grow beyond0.7, the fraction of trailing strand extensions that give rise to asignal of 0.85 or greater 4 standard deviations beyond the mean is lessthan 0.01%. Thus an out-of phase signal arising from a single-baseextension on one of the three sets of trailing strands should bedistinguishable from the in-phase signal with accuracy so long as theout-of-phase signal does not grow beyond −70% of the in-phase signal.

The above discussion assumes that all the extension events correspond tosingle base extensions. However, multiple single-base repeats are commonin DNA sequences, thus one must consider the situation where theout-of-phase signal can be M times larger than that for a single baseextension, where M is the repeat number. For example, if the populationof one of the three sets of out-of-phase strands has grown to 20% of theleading strand population, at which level the in-phase and out-of-phasesignals can readily be distinguished for a single base extension, thenif this set of out-of-phase strands encounters a 5-base repeat, e.g.AAAAA, the signal for that repeat becomes identical in magnitude to thatfor a single base extension on the in-phase strands. Real-time computermonitoring of the extension signals permits discrimination against suchrepeat-enhanced out-of-phase signals, for example, by implementinglinear and/or nonlinear auto-regressive moving average (ARMA) schemes.The essential points here are as follows (i) the out-of-phase strandsare those that are trailing the majority strands as a result ofextension failure; misincorporation events which could produce leadingerror strands have the effect of shutting down further extension on theaffected strands and so do not give rise to significant out-of-phaseerror signals; (ii) there is always only one population of trailingstrands regardless of where the extension failure occurred; all theprimer strands in this population have been extended to the same pointwhich trails the leading strand sequence by 1, 2 or 3 bases; and (iii)because the leading strands have always previously traversed thesequence subsequently encountered by the trailing strands, the sequenceat least 1 base beyond the 3′ terminus of the trailing strands is alwaysknown and allows prediction of exactly whether, and by how much, thesetrailing strands will extend for any nucleotide supplied, by simulating,in a computer for example, the effect of an extension failure at anypoint in the known sequence upstream of the position to which theleading strands have advanced.

On each incorporation trial, in addition to any possible correctextension signal for the leading strands, there may also be an errorsignal corresponding to extension of the trailing strands. For example,let us assume that the trailing strand population has grown as large as20% of the leading strand population. The size of this population can bemonitored by detecting the incorporation signal when the trailingstrands extend and the leading strands do not. Assume that the leadingstrand population has just traversed a single base repeat region on thetemplate, for example AAAAA, and incorporated onto the primer thecomplementary T repeat: TTTTT. The trailing strands will not traversethis same AAAAA repeat for at least a complete cycle of the four probingnucleotides, until the next time the strands are probed with dTTP.Knowing the size of the trailing strand population from the amplitude ofits incorporation signals, determined at any point where the leadingstrands do not extend but the trailing strands do, the signal to beexpected from the trailing strand population due to the TTTTTincorporation can be calculated precisely. If the trailing strandpopulation is ⅕ as large as the leading strand population, for example,this signal will mimic incorporation of a single T on the leading strandpopulation. In the absence of the computer-aided monitoring methoddiscussed here, such a false signal would give rise to a drasticsequence error.

FIGS. 14A and 14B demonstrate how data would appear for a sequence:[CTGA] GAA ACC AGA AAG TCC [T] (SEQ ID NO: 1), probed with a dNTP cycle:CAGT, close to the primer where no extension failure has occurred (FIG.14A) and well downstream (FIG. 14B) at a point where 60% of the strandshave undergone extension failure and are producing out-of-phase signals,and misincorporation has shut down extension on 75% of all strands. Thereadouts shown start at the second G in the sequence (beyond the [CTGA]sequence in parentheses) and end at the last C (before the [T] inparentheses). The digital nature of the signal in FIG. 14A and also theamplitude scale should be noted. In FIG. 14B, the signal for a singlebase extension has been reduced by 60%, from 1.0 to 0.4 due to theextension failure strands, and by a further factor of 4 to 0.1 due tomisincorporation and the resulting 75% signal loss. However, added tothe correct extension signals are signals due to the out-of-phaseextension of the trailing strands. At first sight, the readout iscompletely different from the correct readout shown in FIG. 14A, due tothe superposition of signals produced when the trailing strandsencounter the sequence previously traversed by the leading strands.Particularly large errors arise whenever the trailing strand populationencounters the AAA repeats. For example, the second T probe yields asignal amplitude corresponding to an AAAAA repeat instead of the correctsingle A, the third G probe gives a signal corresponding to CCC when infact there is no C at this point in the leading strand sequence, thefourth T probe reads 4 A's when the correct sequence has none (thetrailing strands encounter the second AAA repeat). However, because thesequence from the leading strands is known, the false signals arisingfrom the trailing strands can be predicted and subtracted from the totalsignal to obtain the correct sequence readout. This is shown in FIG.14C, where the signals arising from the trailing strands are coded bydifferent shading from the leading strand signal. Because the signalsdue to the trailing strands can be predicted, the error signals can besubtracted to obtain the correct digital sequence readout shown in FIG.14D. It should be noted that the data in FIG. 14D are now identical tothose in FIG. 14A, and yield the correct sequence readout for theleading strands, the only difference being that the overall intensity isreduced due to the assumed loss of signal due to misincorporation andextension failure, the latter populating the trailing strands. In otherwords, by keeping track of the sequence in a computer the effect is asthough one could directly visualize the different contributions asdepicted on the plot in FIG. 14C. Therefore, it is possible to predictfor any probe nucleotide event exactly what the signal from the trailingstrand population should be, and subtract this error signal from themeasured signal to arrive at a true digital signal representative of thesequence of the leading strand population, which is the desired result.

Given the ability to compute and subtract any trailing strand signals asdiscussed, the accuracy with which nucleotide incorporation ornon-incorporation on the leading strands can be sensed is limited, notby the absolute size of the trailing strand signal, but instead by thenoise on those signals. For example, assume that the signal for asingle-base extension of a trailing strand population equal to 20% ofthe leading strand population is 0.2±0.05. If the trailing strandsencounter a 5-base repeat, the resulting signal would be identical inamplitude to that produced by a single-base extension of the leadingstrands, but this signal could be subtracted from the observed signal toyield either a signal resulting from a leading strand extension, or anull signal corresponding to no extension of the leading strands.Assuming that the noise is purely statistical and therefore is reducedin proportion to the square root of the signal amplitude, for a 5-baseextension of the trailing strands or a single extension of the leadingstrands the signal would be 1±(0.05×45), i.e. 1±0.11, because thestatistical noise on a set of added signals grows as the square root ofthe number of signals. One can subtract from this value a correctionsignal which is much more accurately known because the trailing strandsignal has been repeatedly measured yielding better statistics on thisvalue. It is assumed that the uncertainty in the correction signal isnegligible. For no extension of the leading strands, the resultingdifference signal would be 0±0.11, whereas a single extension of theleading strands would yield a difference signal of 1±0.11; the twosignals are distinguishable with better than 99.9% accuracy.

The example given here is an extreme case: in fact, the extensionfailure can be corrected at any point, so that it will be possible tominimize the trailing strand population below a level where it wouldproduce signals that make the leading strand sequence uncertain.

There are additional advantages to the computer-aided monitoring methodproposed. First, the signals from the trailing strands serve as anadditional check on the leading strand sequence. Second, the trailingstrand population could be allowed to surpass the leading strandpopulation in magnitude. Without computer-aided monitoring, readoutwould have to cease well before this point, however, with computer-aidedmonitoring, readout can continue, now using the trailing strands ratherthan the leading strands to reveal the sequence. Thus, the strandpopulation that trails due to only one extension failure now becomes theleading strand population for the purposes of computer aided monitoring.This allows readout to continue until further complications arise fromthe occurrence of 2 extension failures on the same strand, producing anew trailing strand population which can be tracked in the same way asthe single failure strands, while the population of strands that haveundergone no error failure diminishes to the point where it contributesno detectable signal.

Optimization of reagents, enzyme and reaction conditions should allowmisincorporation probabilities below 1%, and extension failureprobabilities as low as 0.1%. The computer aided monitoring method ofthe present invention additionally provides a means for healing thetrailing strand population by selectively extending this population sothat it is again synchronous with the leading strands. For example,given a dNTP probe cycle of GCTA, and a template sequence (beyond the 3′end of the primer) of:

(SEQ ID NO: 5) ......GTGCAGATCTG ...

and assuming that when dCTP is in the reaction chamber, the polymerasefails to incorporate a C in some fraction of the primer strands, thefollowing results:

(SEQ ID NO: 5) Template ......GTG CAG ATC TG ... Main strands ......C(SEQ ID NO: 5) Template ......GTG CAG ATC TG Failure strands ......

At the end of the first cycle, the main strands have extended by . . .CA, while the failure strand has not advanced. After one more completecycle, the main strand extension is . . . CAC and the failure strand nowreads . . . CA, i.e. now just one base out of phase.

(SEQ ID NO: 5) Template ......GTG CAG ATC TG ... Main strands ......CAC(SEQ ID NO: 5) Template ......GTG CAG ATC TG Failure strands ......CA

Because the phase lag arises from the repeating interaction of the probecycle sequence with the template sequence, the unchanged probe cycle cannever have the correct sequence to resynchronize the strands. Instead,if the probe cycle is unchanged, and if no further extension failuresoccur, the phase lag for a given failure strand oscillates perpetuallybetween 1 and 3 bases, counting single base repeats as one base for thispurpose. However because the leading strand sequence up to the lastextension is always known, one can determine the effect of introducingan extension failure at some upstream position. It should be noted thatan extension failure introduced at any arbitrary upstream position, orany base type, always produces the same phase lag because the effect ofan extension failure is to cause extension of the affected strand to lagby one complete dNTP cycle. Thus, it is possible to alter the probecycle sequence, for example to probe with a C, instead of a G, after thelast A in the sequence discussed above. The failure strand would advancewhile the main strands did not and the phase lag would heal. In yetanother embodiment the dNTP probe cycle may be reversed whenever thephase lag shrinks to only 1 base. Whenever the phase difference declinesto a single base, or repeats of a single base, then simply reversing theprobe cycle sequence always resynchronizes the strands.

FIG. 15 shows how a leading strand population arising from incorrectextension of a fraction of primer strands due to nucleotide impuritiescan adversely affect the signals from the main population. Using thesame template sequence as before: [CTGA] GAA ACC AGA AA GTC C [TC AGT](SEQ ID NO: 6) and the same probe cycle: CAGT, the effect of a leadingstrand population which is 20% of the main strand population can besimulated and 2 bases ahead of the main strands at the time the mainstrand sequence begins to be read. The leading strands have alreadyextended by -C TTT. The first C probe extends the main primer strands byone base complementary to the first G in the sequence giving a singlebase extension signal of 1. The first G extends the leading strands by-GG- complementary to the -CC- repeat, giving a signal of 0.4. Greaterambiguity arises when the leading strands encounter the secondAAA-repeat at the second T probe, increasing the main strand signal fromthe correct value for a single base extension to 1.6. In the absence offurther information, this value will be ambiguous or may be interpretedas a 2-base repeat.

Correction for these ambiguities comes from the fact that the correctsequence of the main strands is read following the leading strand read.In general, a large multiple repeat which can give an error signal whenencountered by the leading strands will subsequently give a largersignal when encountered by the main strands, and superimposed on thiscorrect signal will be a leading strand signal for which there are threepossibilities: (i) zero signal: the leading strands do not extend; (ii)small signal that does not create ambiguity—the leading strands extendby a single base or a repeat number small enough not to simulate anadditional base extension of the main strands; (iii) large signal; theleading strands encounter a second large repeat. By monitoring the mainstrand sequence, it is possible at each extension to retroactivelyestimate the effects of a leading strand population and subtract suchsignals from the main strand signals to arrive at a correct sequence. Inthe case where the leading strands encounter a repeat large enough tocreate ambiguity in the sequence, even if the leading strandssubsequently encounter a second or third large repeat when the mainstrands encounter the first repeat, the main strands will eventuallytraverse the same region to give sufficient information to derive thecorrect sequence. In other words, the sequence information at any pointis always overdetermined—the signal for any given extension is alwaysread twice, by the leading strands and the main strands, and so yieldssufficient information to determine both the correct sequence and thefractional population of the leading strands, which are the two piecesof information required.

Because the sequence of the leading strand population produced by impurenucleotides cannot be known until it is subsequently traversed by themain strands, one cannot know what dNTP probe cycle would act to extendthe main strands while not extending the leading strands, as was thecase for a trailing strand population produced by extension failure.However, as with trailing strands, the gap between the leading and mainstrands oscillates perpetually between one and three bases, and can bereconnected by reversing the dNTP probe sequence whenever the gapbetween the leading and main strands shrinks to a single base. Althoughit cannot be known when this single base gap occurs, the dNTP probesequence can be reversed at regular intervals. Trials indicate that sucha process ultimately reconnects approximately ⅔ of the leading strands.The statistics for this process are as follows.

Statistically, because the gap between the main and leading strands canbe there is a ⅓ probability that the leading strand population will haveonly a 1. any time the cycle is reversed. The 1-base phase differencewill always be h reversal. Another ⅓ of the time the leading strands are2 bases ahead at th reversed. For the next probing base the followingpossibilities exist:

Lead strand Main strand 0 0 No extension on either strand: Prob. 3/4 ×3/4 = 9/16 +1 0 Phase lag increases: Prob. 1/4 × 3/4 = 3/12 +1 0 Bothstrands advance: Prob. 1/4 × 1/4 = 1/16 0 +1 Phase lag decreases: Prob.3/4 × 1/4 = 3/12 Phase lag stays at 2: Number of chances = 10/16 Phaselag decreases Number of chances = 3/12 Phase lag increases Number ofchances = 3/12

So the chance of making a 2-base gap worse is ( 3/12)/( 10/16+ 3/12)=28%three gap sizes: 1-base gap heals (33% of population); 2-base gap getswore only ⅓ of gaps are 2 base, so 9% total get worse; 3-base gap alsogets wo again 9% overall effect. In sum, 33% heal at a given reversal,18% lose at a the remaining 50% are unchanged. Even assuming the 18% arepermanent gap increased to a 3 base gap can still rejoin), at eachsubsequent reversal I strands are healed, which are unchanged by theprevious reversal, as follow

Reversal # Fraction of gaps healed 1 33% 2 17% 3  9% 4 4.5%  5 2.5%  6 1% Total ~67%  

Therefore, repeated reversal of the dNTP probe cycle can reduce by ⅔ thephase signals due to incorrect extension by nucleotide impurities, orrandom effectively increasing the read length when limited by eithereffect by a factor

Although the invention has been described herein with reference tospecific embodiments, many modifications and variations therein willreadily occur to those skilled in the art. Accordingly, all suchvariations and modifications are included within the intended scope ofthe invention.

1-32. (canceled)
 33. A method for phase error compensation, comprising:(a) providing a plurality of template nucleic acid strands; (b) addingdeoxynucleoside triphosphates serially to the plurality of templatenucleic acid strands to perform nucleotide incorporation reactions; (c)detecting incorporation signals having an amplitude corresponding to anumber of nucleotide incorporations resulting from the addeddeoxynucleoside triphosphates; and (d) compensating for phasing errorsdue to one or more out-of-phase template nucleic acid strand populationsby altering an order of addition of the deoxynucleoside triphosphates.34. The method of claim 33, wherein the plurality of template nucleicacid strands are hybridized to a primer.
 35. The method of claim 33,wherein the one or more out-of-phase template nucleic acid strandpopulations comprises a trailing fraction of the template nucleic acidstrands that are out of phase relative to an in-phase fraction of thetemplate nucleic acid strands because of extension failures.
 36. Themethod of claim 35, wherein compensating for phasing errors comprisesmonitoring the trailing fraction of the template nucleic acid strands todetermine where and how to alter the order of addition of thedeoxynucleoside triphosphates so as selectively to extend the trailingfraction of the template nucleic acid strands to bring them back intophase with the in-phase fraction of the template nucleic acid strands.37. The method of claim 35, wherein compensating for phasing errorscomprises reversing a cycle of addition of the deoxynucleosidetriphosphates at arbitrary intervals to bring back into phase aboutone-third of the trailing fraction of the template nucleic acid strands.38. The method of claim 33, wherein the one or more out-of-phasetemplate nucleic acid strand populations comprise a leading fraction ofthe template nucleic acid strands that are out of phase relative to anin-phase fraction of the template nucleic acid strands because ofincorrect extensions.
 39. The method of claim 38, wherein compensatingfor sequencing errors comprises reversing a cycle of addition of thedeoxynucleoside triphosphates at arbitrary intervals to bring back intophase about two-thirds of the leading fraction of the template nucleicacid strands.
 40. The method of claim 35, wherein the one or moreout-of-phase template nucleic acid strand populations further comprise aleading fraction of the template nucleic acid strands that are out ofphase relative to an in-phase fraction of the template nucleic acidstrands because of incorrect extensions.
 41. The method of claim 40,wherein compensating for sequencing errors comprises reversing a cycleof addition of the deoxynucleoside triphosphates at arbitrary intervalsto bring back into phase about two-thirds of the leading fraction of thetemplate nucleic acid strands.
 42. A method for increasing read lengthduring sequencing, comprising: (a) providing a plurality of templatenucleic acid strands; (b) adding deoxynucleoside triphosphates to theplurality of template nucleic acid strands to perform nucleotideincorporation reactions according to a serial addition cycle; (c)detecting incorporation signals having an amplitude corresponding to anumber of nucleotide incorporations resulting from the addeddeoxynucleoside triphosphates; and (d) compensating for phasing errorsdue to template nucleic acid strands having undergone extension failuresby arbitrarily reversing the addition cycle.
 43. A method for increasingread length during sequencing, comprising: (a) providing a plurality oftemplate nucleic acid strands; (b) adding deoxynucleoside triphosphatesto the plurality of template nucleic acid strands to perform nucleotideincorporation reactions according to a serial addition cycle; (c)detecting incorporation signals having an amplitude corresponding to anumber of nucleotide incorporations resulting from the addeddeoxynucleoside triphosphates; and (d) compensating for phasing errorsdue to template nucleic acid strands having undergone incorrectextensions by arbitrarily reversing the addition cycle.